U.S. patent number 5,256,558 [Application Number 07/514,816] was granted by the patent office on 1993-10-26 for gene encoding plant asparagine synthetase.
This patent grant is currently assigned to The Trustees of Rockefeller University. Invention is credited to Gloria M. Coruzzi, Fong-Ying Tsai.
United States Patent |
5,256,558 |
Coruzzi , et al. |
October 26, 1993 |
Gene encoding plant asparagine synthetase
Abstract
The identification and cloning of the gene(s) for plant
asparagine synthetase (AS), an important enzyme involved in the
formation of asparagine, a major nitrogen transport compound of
higher plants is described. Expression vectors constructed with the
AS coding sequence may be utilized to produce plant AS; to engineer
herbicide resistant plants, salt/drought tolerant plants or
pathogen resistant plants; as a dominant selectable marker; or to
select for novel herbicides or compounds useful as agents that
synchronize plant cells in culture. The promoter for plant AS,
which directs high levels of gene expression and is induced in an
organ specific manner and by darkness, is also described. The AS
promoter may be used to direct the expression of heterologous
coding sequences in appropriate hosts.
Inventors: |
Coruzzi; Gloria M. (New York,
NY), Tsai; Fong-Ying (New York, NY) |
Assignee: |
The Trustees of Rockefeller
University (New York, NY)
|
Family
ID: |
26995216 |
Appl.
No.: |
07/514,816 |
Filed: |
April 26, 1990 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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347302 |
May 3, 1989 |
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Current U.S.
Class: |
435/252.33;
435/252.3; 435/320.1; 536/23.2; 536/24.1 |
Current CPC
Class: |
C07K
16/40 (20130101); C12N 9/93 (20130101); C12N
15/8209 (20130101); C12N 15/8273 (20130101); C12N
15/8227 (20130101); C12N 15/8237 (20130101); C12N
15/8222 (20130101) |
Current International
Class: |
C07K
16/40 (20060101); C12N 15/82 (20060101); C12N
9/00 (20060101); C12N 005/10 (); C12N 001/19 ();
C12N 001/21 (); C12N 015/52 () |
Field of
Search: |
;435/91,172.3,240.1,252.3,252.33,254,255,183,320.1 ;536/27
;935/33,34,35 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Wilson (1971), Botany (Holt, Rinehart, & Winston, NY), pp.
447-451. .
Joy et al. (1983), Plant Physiology, vol. 73, pp. 165-168. .
Ray et al. (1984), Gene, vol. 30, pp. 1-9. .
Andrulis et al. (1987), Mol. Cell. Biol., vol. 7, pp. 2435-2443.
.
Lea and Fowden (1975), Proc. R. Soc. Lond. B, vol. 192, pp. 13-26.
.
Pharmacia, "Cloning Vectors", Pharmacia Catalog, 1989. .
Humbert et al. (1980), Journal of Bact., vol. 142, pp. 212-220.
.
Davidson et al. (1983), Proc. Natl. Acad. Sci., vol. 80, pp.
6897-6901. .
Tingey et al., 1987, EMBO Journal 6: 1-9. .
Andrulis et al., 1985, J. Biol. Chem. 260(12): 7523-7527. .
Herrera-Estrella et al., 1984, Nature 310: 115-120. .
Maniatis et al., 1982, "Molecular Cloning," Cold Spring Harbor
Library, pp. 295-299. .
Rognes, 1975, Phytochen 14:1975. .
Ramnefjell & Rognes, 1981, Suppl. Plant Physiol., Abstr. 244.
.
Huber & Streeter, 1985, Plant Science 42:9-17. .
Shelp et al., 1984, Plant science Letters 36:225-230. .
Huber & Streeter, 1984, Plant Physiol. 74:605-610. .
Loyola-Vargas et al., 1988, J. Plant Physiol. 132:289-293. .
Roth & Lark, 1984, Theor. Appl. Genet. 68:421-431. .
Hongo et al., 1978, Biochim. Biophys. Acts 522:258-266. .
Milman et al., 1979, Biochem. J. 181:51-59. .
Markin & Schuster, 1979, Biochem. Biophys. Res. Commun.
88(2):583-588. .
Hongo & Sato, 1981, Anal. Biochem. 114:163-166. .
Hongo & Sato, 1983, Biochem. Biophys. Acta 742:484-489. .
Pfeiffer et al., 1986, J. Biol. Chem. 261 (4):1914-1919. .
Pfeiffer et al., 1987, J. Biol. Chem. 262(24):11565-11570. .
Arfin et al., 1983, Somatic Cell Genet. 9(5):517-531. .
Smith et al., 1984, Cytogen. Cell Genet. 37(1-4) 1985-1986. .
Van Heeke & Schuster, 1989, J. Biol. Chem. 264(10), 5503-5509.
.
Jones, 1978, J. Bacteriol. 134:200-207. .
Ramos & Wiame, 1980, Eur. J. Biochem. 118:373-377. .
Nakamura, 1981, Nuc. Acids. Res. 9(18):4669-4697. .
Scofield & Schuster, 1988, J. Cell Biol. 107:535(a), Abstr.
3023. .
Waye et al., 1983, J. Mol. Appl. Genet. 2:69-82. .
Cartier et al., 1987, Mol. Cell. Biol. 71(5):1623-1628. .
Tsai & Coruzzi, 1990, EMBO J. 9(2):323-332..
|
Primary Examiner: Schwartz; Richard A.
Assistant Examiner: Vogel; Nancy T.
Attorney, Agent or Firm: Pennie & Edmonds
Government Interests
The invention described herein was supported in whole or in part by
a grant from the National Institute of Health, and the United
States Department of Energy.
Parent Case Text
The present application is a continuation in part of application
Ser. No. 07/347,302 filed May 3, 1989, now abandoned.
Claims
What is claimed is:
1. An isolated nucleotide sequence encoding plant asparagine
synthetase which is capable of selectively hybridizing to the
nucleotide sequence of FIG. 2a or 2b, or which encodes
substantially the same amino acid sequence of FIG. 2a or 2b.
2. A substantially pure nucleotide sequence encoding plant
asparagine synthetase comprising the nucleotide sequence
substantially as depicted in FIG. 2A from nucleotide residue number
94 to nucleotide residue number 1851.
3. A substantially pure nucleotide sequence encoding plant
asparagine synthetase comprising the nucleotide sequence
substantially as depicted in FIG. 2B from nucleotide residue number
102 to nucleotide residue number 1850.
4. A recombinant DNA molecule containing a nucleotide sequence
encoding plant asparagine synthetase, which is capable of
selectively hybridizing to the nucleotide sequence of FIG. 2a or
2b, or which encodes substantially the same amino acid sequence of
FIG. 2a or 2b.
5. The recombinant DNA molecule according to claim 4 in which the
nucleotide sequence encoding plant asparagine synthetase comprises
the nucleotide sequence substantially as depicted in FIG. 2A from
nucleotide residue number 94 to 1851.
6. The recombinant DNA molecule according to claim 4 in which the
nucleotide sequence encoding plant asparagine synthetase comprises
the nucleotide sequence substantially as depicted in FIG. 2B from
nucleotide residue number 102 to 1850.
7. Recombinant plasmid pTZ18U/cAS1.
8. Recombinant plasmid pTZ19U/cAS201.
9. Recombinant plasmid pTZ18U/gAS1.a.
10. Recombinant plasmid pTZ18U/gAS2.a.
11. Recombinant plasmid pTZ19U/cAS801.
12. A cultured cell containing a recombinant nucleotide sequence
encoding plant asparagine synthetase which is capable of
selectively hybridizing to the nucleotide sequence of FIG. 2a or
2b, or which encodes substantially the same amino acid sequence of
FIG. 2a or 2b.
13. The cultured cell according to claim 12 in which the nucleotide
sequence encoding plant asparagine synthetase comprises the
nucleotide sequence substantially as depicted in FIG. 2A from
nucleotide residue number 94 to 1851.
14. The cultured cell according to claim 12 in which the nucleotide
sequence encoding plant asparagine synthetase comprises the
nucleotide sequence substantially as depicted in FIG. 2B from
nucleotide residue number 102 to 1850.
15. Escherichia coli XL1 transformed with pTZ18U/cAS1 as deposited
with the NRRL and assigned accession number B-18487.
16. Escherichia coli XL1 transformed with pTZ19U/cAS201 as
deposited with the NRRL and assigned accession number B-18486.
17. Escherichia coli XL1 transformed with pTZ19U/cAS801 as
deposited with the NRRL and assigned accession number B-18649.
18. A cultured cell containing a recombinant genomic nucleotide
sequence encoding exons for plant asparagine synthetase, which is
capable of selectively hybridizing to the nucleotide sequence of
FIG. 2a or 2b, or which encodes substantially the same amino acid
sequence of FIG. 2a or 2b.
19. A cultured cell containing a recombinant genomic nucleotide
sequence for asparagine synthetase as contained in
pTZ18U/gAS1.a.
20. A cultured cell containing a recombinant genomic nucleotide
sequence for asparagine synthetase as contained in a
pTZ18U/gAS2.a.
21. Escherichia coli XL1 transformed with pTZ18U/gAS1.a as
deposited with the NRRL and assigned accession number B-18492.
22. Escherichia coli XL1 transformed with pTZ18U/gAS2.a as
deposited with the NRRL and assigned accession number B-18493.
23. A cultured cell containing a recombinant nucleotide sequence
encoding plant asparagine synthetase controlled by a second
nucleotide sequence that regulates gene expression in the cultured
cell, so that the cultured cell produces plant asparagine
synthetase, in which the recombinant nucleotide sequence is capable
of selectively hybridizing to the nucleotide sequence of FIG. 2a or
2b, or encodes substantially the same amino acid sequence of FIG.
2a or 2b.
24. The cultured cell according to claim 23 in which the nucleotide
sequence encoding plant asparagine synthetase comprises the
nucleotide sequence substantially as depicted in FIG. 2A from
nucleotide residue number 94 to 1851.
25. The cultured cell according to claim 23 in which the nucleotide
sequence encoding plant asparagine synthetase comprises the
nucleotide sequence substantially as depicted in FIG. 2B from
nucleotide residue number 102 to 1850.
26. A substantially pure plant asparagine synthetase promoter
comprising the nucleotide sequence substantially as depicted in
FIG. 10 from nucleotide residue number -558 to +11.
27. A substantially pure plant asparagine synthetase promoter
comprising the nucleotide sequence substantially as depicted in
FIG. 10 from nucleotide residue number -2376 to +11.
28. A substantially pure plant asparagine synthetase promoter
comprising the nucleotide sequence substantially as depicted in
FIG. 12 from nucleotide residue number 1 to 1031.
29. A recombinant DNA molecule containing an inducible plant
asparagine synthetase promoter which is capable of selectively
hybridizing to the nucleotide sequence of FIG. 10 or FIG. 12.
30. A recombinant DNA molecule containing a plant asparagine
synthetase promoter which is inducible by dark and is capable of
selectively hybridizing to the nucleotide sequence of FIG. 10 or
FIG. 12.
31. The recombinant DNA molecule according to claim 29 in which the
plant asparagine synthetase promoter comprises the nucleotide
sequence substantially as depicted in FIG. 10 from nucleotide
residue number -558 to +11.
32. The recombinant DNA molecule according to claim 29 in which the
plant asparagine synthetase promoter comprises the nucleotide
sequence substantially as depicted in FIG. 10 from nucleotide
residue number -2376 to +11.
33. The recombinant DNA molecule according to claim 29 in which the
plant asparagine synthetase promoter comprises the nucleotide
sequence substantially as depicted in FIG. 12 from nucleotide
residue number 1 to 1031.
Description
TABLE OF CONTENTS
1. Introduction
2. Background Of The Invention
2.1. Mammalian, Bacterial And Yeast Asparagine Synthetases
2.2. Plant Asparagine Synthetase
3. Summary Of The Invention
3.1. Definitions
4. Description Of The Figures
5. Description Of The Invention
5.1. Plant Asparagine Synthetase Coding Sequence
5.2. Expression Of Plant Asparagine Synthetase
5.2.1. Construction Of Expression Vectors Containing The Plant AS
Coding Sequence
5.2.2. Identification of Transfectants Or Transformants Expressing
The Plant AS Gene Product And Isolation Of Plant AS
5.3. Uses Of Plant Asparagine Synthetase Gene and Gene Products
5.3.1. Production Of Antibodies That Define And/Or Inhibit Plant
AS
5.3.2. Development Of New Herbicides
5.3.3. Development Of Herbicide Resistant Plants And Stress
Tolerant Plants
5.3.4. Identification Of Agents That Synchronize Plant Cells In
Culture
5.4. The Plant Asparagine Synthetase Promoter
6. Example: cDNA Cloning Of Plant Asparagine Synthetase
6.1. Materials and Methods
6.1.1. Isolation Of Plant AS1 cDNAs
6.1.2. Synthesis Of Full Length AS1 cDNA
6.1.3. Isolation Of Plant AS Genomic Clones
6.1.4. Isolation Of Plant AS2 cDNAs
6.1.5. DNA And RNA Analyses
6.2. Results
6.2.1. Isolation Of Two Classes Of Pea AS cDNA Clones
6.2.2. pcAS1 And cAS2 Represent Homologous AS mRNAs Which Encode
Distinct AS Polypeptides
6.2.3. AS1 And AS2 Are Encoded By Single Nuclear Genes In The Pea
Genome
6.2.4. Photophobic Accumulation Of AS1 mRNA In Leaves
6.2.5. Both AS1 and AS2 mRNAs Are Expressed At High Levels During
Developmental Contexts Involving Increased Nitrogen Transport
6.3. Discussion
7. Example: The AS Promoter
7.1. Dark-Induced Accumulation Of AS mRNA Occurs In All Organs
Tested
7.2. The Dark-Induced Expression Of AS1 Gene Is Not Regulated By
Circadian Rhythm
7.3. The Dark-Induced Expression Of AS Genes In Pea Leaves Is
Regulated At The Level Of Transcription
7.4. The 569 Base Pair Fragment Of The AS1 Promoter Is Sufficient
To Drive GUS Expression In Transgenic Plants
7.5. AS1 And AS2 Genes Contain Conserved Sequence In Their
Promoters
8. Deposit Of Microorganisms
1. INTRODUCTION
The present invention relates to the cloning and expression of
plant asparagine synthetase, an important enzyme involved in the
formation of asparagine, a major nitrogen transport compound of
higher plants. Recombinant DNA techniques are used to construct
cDNA clones of plant asparagine synthetase. These clones may be
used to construct expression vector/host systems that produce plant
asparagine synthetase which, in turn, can be used for a variety of
purposes including the generation of antibodies that define plant
asparagine synthetase. The clones can also be used to overproduce
wild type or altered forms of asparagine synthetase in order to
engineer herbicide resistant plants, salt/drought tolerant plants,
and pathogen resistant plants; as a dominant selectable marker;
and/or to select for novel herbicides or compounds useful as agents
which synchronize plant cells in culture.
The invention also relates to the promoter for plant asparagine
synthetase which is induced by darkness and directs high levels of
transcription.
2. BACKGROUND OF THE INVENTION
In higher plants, nitrogen assimilated from the soil must be
incorporated into organic form for transport to the growing plant.
The amides, asparagine and glutamine, are the two major nitrogen
transport compounds in most higher plants (Sieciechowicz et al.,
1988, Phytochemistry 27:663-671). These amides function to deliver
nitrogen to and from plant organs at various stages of plant
development. Since the production of asparagine and glutamine for
nitrogen transport is crucial to plant growth, the enzymes involved
in their synthesis are targets for herbicide action. Much is known
about plant glutamine synthetase (GS) as an enzyme, a
herbicide-target and as a gene family. In contrast, very little is
known about plant asparagine synthetase (AS) at the biochemical
level, at the herbicide-target level and nothing is known about the
AS gene(s) in higher plants.
Asparagine is the major nitrogen transport compound which is
synthesized when a plant is faced with excess ammonia rather than
nitrate. During normal plant growth, conditions of ammonia excess
arise when (1) plants are treated with externally applied
fertilizers; (2) plants develop nitrogen-fixing root nodules; (3)
the seed storage proteins of cotyledons are deaminated during
germination; and (4) senescing plants are mobilizing nitrogen for
seed formation. In certain species asparagine can account for up to
86% of transported nitrogen in the above contexts. See, Lea &
Miflin, 1980, Transport And Metabolism Of Asparagine And Other
Nitrogen Compounds Within The Plant, in The Biochemistry Of Plants,
Volume 5, Ed. Miflin, Acad. Press, N.Y., Ch. 16.
There are several reasons why asparagine is preferred as a nitrogen
transport/storage compound compared to glutamine in situations
requiring the assimilation and transport of large amounts of
nitrogen. Asparagine contains a high N:C ratio (2N:4C) compared to
glutamine (2N:5C) which makes asparagine a more economical nitrogen
transport/storage compound compared to glutamine. Asparagine is
also a preferred compound for nitrogen transport/storage because it
is relatively inert compared to glutamine. Because glutamine is
such an active metabolite which donates the amide nitrogen to a
large number of substrates, over-production of glutamine could
seriously upset plant metabolism. Storage of high levels of
nitrogen containing compounds is extremely important in vegetative
structures such as fruits where asparagine stored in vacuoles
serves as the primary source of nitrogen for seed storage protein
synthesis in developing seeds.
Despite the crucial role of asparagine synthetase during plant
development, very little is known about the AS enzyme in higher
plants. Years of labor intensive biochemical investigations on
plant AS have failed to produce a homogeneous enzyme preparation.
The inability to purify AS biochemically is due, in part, to the
fact that AS is extremely unstable in vitro in these partially
purified extracts. In addition, AS activity is difficult to detect
in partially purified extracts due to contaminating asparaginase
activity, and due to the presence of specific non-protein
inhibitors of AS activity.
2.1. MAMMALIAN, BACTERIAL AND YEAST ASPARAGINE SYNTHETASE
In contrast to the difficulties encountered in studies of plant AS
enzyme, researchers have had more success studying AS in other
systems. Methods for the purification of mammalian AS involve
fractionation of the enzyme from the liver or pancreas of animals
whose diets are altered to increase AS production in these organs.
Methods involving ammonium sulfate frationation, ion exchange
chromotography, affinity chromatography, and sucrose gradient
centrifugation, have been reported for the partial purification of
the mammalian AS enzyme. See, for example, Hongo, et al., 1978,
Biochim. Biophys. Acta 522:258-266; Milman et al., 1979, Biochem.
J. 181:51-59; Markin & Schuster, 1979, Biochem. Biophys. Res.
Commun. 88(2):583-588; Hongo & Sato, 1981, Anal. Biochem.
114:163-166; and Hongo & Sato, 1983, Biochim. Biophys. Acta
742:484-489. Some researchers have used the partially purified
mammalian AS preparation to generate monoclonal antibodies that
define the enzyme in order to study mammalian AS (Pfeiffer et al.,
1986, J. Biol. Chem. 261(4):1914-1919; Pfeiffer et al., 1987, J.
Biol. Chem. 262 (24):11565-11570).
Somatic cell genetic studies assigned the structural gene for human
AS to chromosome 7 (Arfin et al., 1983, Somatic Cell Genet.
9(5):517-531; Smith et al., 1984, Cytogen. Cell Genet.
37(1-4):585-586). Ultimately, cDNAs encoding mammalian AS were
cloned (Ray et al., 1984, Gene 30:1-9; Andrulis et al., 1987, Mol.
Cell Biol. 7:2435-2443); and very recently expressed in bacteria
(Van Heeke & Schuster, 1989, J. Biol. Chem. 264
(10):5503-5509).
Bacteria have two forms of asparagine synthetase (AsnA and AsnB)
(Humbert & Simoni, 1980, J. Bacteriol. 142:212-220; Nakamura,
1981, Nuc. Acids Res. 9(18):4669-4697; Scofield & Schuster,
198S, J. Cell Biol. 107:535a, Abstr. 3023). Bacterial AS has been
cloned and transferred to mammalian cells in a complementation
system, or as a dominant selectable marker (Waye et al., 1983, J.
Mol. Appl. Genet. 2:69-82; Cartier et al., 1987, Mol. Cell. Biol.
71(5):1623-1628).
Yeast also have two genes for AS (ASN1 and ASN2) which have been
defined genetically but have not been isolated or cloned (Jones,
1978, J. Bacteriol. 134:200-207; Ramos & Wiame, 1980, Eur. J.
Biochem. 108:373-377).
2.2. PLANT ASPARAGINE SYNTHETASE
Attempts to purify plant AS using standard biochemical approaches
have been largely unsuccessful. For example, attempted purification
of AS from Lupin cotyledons and wheat seedlings using salt
fractionation, negative absorption on alumina gel and gel
filtration revealed that the enzyme is very labile and sensitive to
high salt concentrations (Lea & Fowden, 1975, Proc. R. Soc.
London B. 192:18-26; Rognes, 1975, Phytochem. 14:1975; Ramnefjell
& Rognes, 1981, Suppl. Plant Physiol., Abstr. 244). Other
schemes developed for purification of AS from soybean root nodules
involving ammonium sulfate precipitation of crude extracts and
chromatography of the supernatant on alumina gel and Reactive Blue
2-cross linked Agarose resulted in a preparation of low yield and
limited stability (Huber & Streeter, 1985, Plant Science
42:9-17).
As a result of the inability to purify plant AS, studies of the
enzyme in plants are largely confined to using assays designed to
detect AS activity in various organs and/or subcellular locations
within the plant during development. See, for example, Joy et al.,
1983, Plant Physiol. 73:165-168; Shelp et al., 1984, Plant Science
Letters 36:225-230; Huber & Streeter, 1984, Plant Physiol.
74:605-610; and Loyola-Vargas et al., 1988, J. Plant Physiol. 132:
289-293.
Unlike mammalian, bacterial and yeast systems, absolutely nothing
is known about AS gene(s) in higher plants. Although auxotrophic
mutants have been reported, the nature of these mutations has not
been elucidated or genetically defined and, indeed, such analysis
appears to be rather complex (Roth and Lark, 1984, Theor. Appl.
Genet. 68:421-431).
3. SUMMARY OF THE INVENTION
The present invention relates to the cloning and expression of
plant asparagine synthetase, an important enzyme involved in the
formation of asparagine--a major nitrogen transport compound of
higher plants. The invention also relates to the plant AS promoter
which may be used to direct the expression of heterologous coding
sequences. The AS promoter directs high levels of expression in
nitrogen fixing root nodules, in cotyledons of germinating seeds,
and dark-induced gene expression in leaves, stems and roots. This
promoter can be used to direct the regulated expression of
heterologous gene sequences in appropriate hosts.
To overcome the major problems encountered in the previous
biochemical studies of plant AS, the present invention employs
molecular techniques to directly isolate cDNA clones encoding plant
AS. DNA sequence analysis of these cDNA clones revealed that plants
contain at least two different AS mRNAs (AS1 and AS2) which encode
homologous but distinct AS polypeptides. Full length cDNAs for
plant AS can be used in a "reverse biochemical" approach to
synthesize and characterize the encoded AS proteins. The ability to
use the cloned plant AS cDNAs to synthesize the purified AS
proteins will allow a characterization of the distinct plant AS
enzyme(s) in terms of physical properties (i.e., substrate
preference, ammonia or glutamine), herbicide sensitivity, and
subcellular localization (i.e., plastid vs. cytosol).
The isolated cDNAs encoding plant AS may also be used to create
herbicide resistant plants and salt/drought tolerant plants,
pathogen resistant plants or to develop novel herbicides or agents
that synchronize plant cells in culture as described in more detail
herein. For example, organisms which express the cloned plant AS
cDNAs can be used to select for mutations in the AS gene(s) which
produce an AS enzyme resistant to known AS inhibitors (for example,
.beta. aspartyl hydroxamate, albizziine, azaserine, etc.).
Expression of such altered AS gene(s) or over-expression of
wild-type AS gene(s) directed by a constitutive or inducible
promoter in transgenic plants can produce plants resistant to known
AS inhibitors. Additionally, over-expression of AS gene(s) in
plants may confer salt/drought tolerance, or pathogen
resistance.
Full length AS cDNA clones may also be used to synthesize highly
purified preparations of the wild-type or altered AS enzyme in
vitro or in vivo (e.g., in Asn.sup.- bacteria, algae, neurospora,
yeast, plant, or animal cells), or as a dominant selectable marker
in any of these systems. In vitro or in vivo synthesized AS can be
used as a substrate in a screen to identify novel herbicidal
compounds, or cell cycle regulatory compounds which selectively
inhibit this important but poorly understood plant enzyme.
3.1. DEFINITIONS
The following terms as used herein, whether in the singular or
plural, shall have the meanings indicated.
______________________________________ Asn = asparagine AS =
asparagine synthetase bp = base pair EDTA = disodium ethylene
diamine tetracetate GS = glutamine synthetase kb = kilobase SDS =
sodium dodecyl sulfate 20 .times. SSC = 175.3 g NaCl and 88.2 g
sodium citrate in 800 ml H.sub.2 O, pH 7.0 (adjusted with 10 N
NaOH), adjusted to 1 liter.
______________________________________
4. DESCRIPTION OF THE FIGURES
FIG. 1. Restriction maps of cDNA clones and genomic clones,
encoding AS1 and AS2. E=EcoRI, S=SstI, Bm=BamHI, Bg=BglII,
H=HincII. E.sup.- represents an EcoRi site in .lambda.cAS301 which
was lost in the process of cloning and selection. Open bars
represent the coding region of each cDNA:
FIG. 1a. pcAS1 (2.2 kb) is a composite full length cDNA constructed
from restriction fragments (a, b and c) of overlapping cDNA clones
(.lambda.cAS907, .lambda.cAS301 and .lambda.cAS305). .lambda.cAS301
and .lambda.cAS305 were isolated via heterologous hybridization to
human AS cDNA (Andrulis et., al., 1987 Mol. Cell Biol.
7:2435-2443), while .lambda.cAS907 was synthesized as described in
the examples.
FIG. 1b. Overlapping cDNA clones pcAS801 and .lambda.cAS201 for AS2
mRNA were used to derive the restriction map of full-length cAS2.
pcAS201 corresponds to a partial cDNA (1.5 kb) for AS2 mRNA (2.2kb)
which was isolated by hybridization to AS2 genomic clone
.lambda.gAS2 (FIG. 1C) which was obtained from a pea genomic
library by cross-hydridization to AS1 cDNAs. .lambda.cAS801 was
synthesized as described in the examples herein.
FIG. 1c and d. Restriction maps of lambda genomic clones
.lambda.gAS1 and .lambda.gAS2 and their respective subcloned
restriction fragments pgAS1.a and pgAS2.a. .lambda.gAS1 is a 17-18
kb SalI restriction fragment containing the pea AS1 gene. The line
below .lambda.gAS1 denotes the 3.2 kb SstI-BamHI fragment subcloned
into pTZ18U, called pgAS1.a. .lambda.gAS2 is a 16-17 kb SalI
fragment containing the pea AS2 gene. The line below .lambda.gAS2
denotes a 5.5 kb BamHI fragment subcloned into pTZ18U, called
pgAS2.a. The shaded bar under .lambda.gAS2 represents the 1.5 kb
EcoRI-BamHI restriction fragment of .lambda.gAS2 which was used to
isolate AS2 cDNAs. Arrows marked "2E" represent two EcoRI
restriction sites which are not definitively mapped. E=EcoRI,
B=BamHI, S=SstI. SalI sites marked correspond to sites derived from
the .lambda.EMBL3 vector (Frischauf et al. 1983, J. Mol. Bio. 170,
827-842; Lycett et al 1985 Nuc. Acids Resh 3:6733-6743).
FIG. 2. Nucleotide sequences of cDNAs encoding pea AS1 and AS2.
FIG. 2a-2j. Nucleotide sequences of cDNAs pcAS1 (FIG. 2a-e) and
cAS2 (FIG. 2f-j) are shown in the mRNA sense. The deduced amino
acid sequence is denoted above the nucleotide sequence in the
standard one letter code. Amino acids are numbered starting with
the first inframe methionine as 1. 3' sequences extending beyond
the pcAS1 cDNA insert (i.e., to residue number 2135 in FIG. 2e)
derived from other overlapping cDNA clones are also shown. The
translation termination codons in each clone are designated as
"*".
FIG. 3 (parts a-d). Amino acid sequence homology of plant AS and
human AS. The deduced amino acid sequences encoded by the Pisum
sativum cDNA clones pcAS1 and cAS2, and human AS cDNA (pH131)
(Andrulis et al., 1987, Mol. Cell Biol. 7:2435-2443) are compared.
Amino acids are denoted in the standard one letter code. Double
dots denote identities between pea AS1 and pea AS2 or the pea AS1
and human AS sequences as shown. Dashes in the amino acid sequences
represent deletions used to maximize homology of AS proteins. Amino
acid alignment is according to "fasta" computer program (Pearson
and Lipman, 1988, Proc. Natl. Acad. Sci., USA, 85:2444-2448).
FIG. 4(parts a and b). Southern blot analysis of AS genes in Pisum
sativum demonstrates that AS1 and AS2 are each encoded by a single
nuclear gene in the pea genome. Pea nuclear DNA was digested with
the following restriction enzymes: S=SstI (lanes 1 and 5), E=EcoRI
(lanes 2 and 6), B=BamHI (lanes 3 and 7), and H=HindIII (lanes 4
and 8), resolved by gel electrophoresis, transferred to
nitrocellulose, and probed with radioactive probes derived from the
coding region of each cDNA pAS1 (FIG. 4A) or cAS2 (FIG. 4B) as
described. In each digestion, a single radioactive band is detected
by the DNA probe which corresponds to a single nuclear gene.
FIG. 5. Northern blot analysis demonstrates that AS1 mRNA
accumulates to high levels in leaves in a "photophobic" fashion;
i.e., AS1 mRNA accumulates in the dark. A gene-specific DNA probe
from the 3, non-coding region of pcAS1 was used to probe AS1 mRNA
(2.2 kb) in RNA from leaves of dark (D) or light (L) treated pea
plants. As a control, mRNAs for chloroplast and cytosolic GS (1.5
and 1.4 kb, respectively) were also detected on the Northern blots
with cDNA probes pGS185 and pGS299 (Tingey et al., 1988, J. Biol.
Chem. 263(20):9651-9657):
FIG. 5a: Total RNA (20 .mu.g) from leaves of peas grown in
continuous white light for 14 days (lane 1), transferred to the
dark for 6 hours (lane 2); or 3 days (lane 3) and back to
continuous white light for 6 hours (lane 4) or 1 day (lane 5).
FIG. 5b: Total RNA (20 .mu.g) from leaves of 10, 17, 24 and 31 day
old plants. Controls were grown in continuous white light for 10,
17, 24 and 31 days ("L" lanes 1, 3, 5, and 7). Dark treated plants
were grown in continuous white light for 7, 14, 21, 28 days and
then transferred to the dark for an additional 3 days ("D" lanes 2,
4, 6, 8).
FIG. 5c: Total RNA (20 g) from leaves of pea plants grown in the
dark for 7 days (lane 1) and transferred to the light for 72 hours
(lane 2).
FIG. 5d. Total RNA (20 .mu.g) from leaves of peas grown in normal
light/dark cycle for 14 days (16 hours light, 8 hours dark) after
which plants were collected during the day (light conditions) (lane
1), transferred to the dark for 4 days (lane 2) and back to
continuous white-light for 24 hours (lane 3).
FIG. 5e: Total RNA (20 g) from leaves of peas grown in the dark for
9 days (lane 1) and subsequently treated with a pulse of red light
then put back to the dark for 3 hours (lane 2), treated with a
pulse of red light followed by a pulse of far-red light then put
back into the dark for 3 hours (lane 3) or transferred to
continuous white light for 8 hours (lane 4).
FIG. 6. Northern blot analysis of AS mRNA accumulation in
cotyledons of germinating seedlings and in nitrogen fixing root
nodules reveal that AS1 and AS2 mRNAs accumulate in contexts of
increased nitrogen transport. Gene specific DNA fragments from the
3' non-coding region of pcAS1 or cAS2 were used to detect AS1 mRNA
(2.2 kb) AS2 mRNA (2.2 kb) on Northern blots. As a control, mRNA
for the .beta.-subunit of mitochondrial ATPase (2.1 kb) was
monitored (Boutry & Chua, 1985, EMBO J. 4(9):2159-2166):
FIG. 6a: Total RNA (20 .mu.g) extracted from cotyledons of pea
seedlings germinated 2 to 18 days (lanes 1-8).
FIG. 6b: RNA extracted from roots as follows: 1 .mu.g
polyadenylated RNA isolated from roots of uninfected pea plants
(lane 1) or nitrogen fixing root nodules (lane 2).
FIG. 7: Dark-Induced expression of AS mRNA occurs independent of
organ-type Northern blot analysis was performed to detect the
effect of the light on steady-state levels of AS mRNA in leaves,
stems and roots. Pea plants were grown in continuous light for 28
days (L, Lanes 1, 3, 5) or grown in continuous light for 25 days
and transferred to the dark for 3 days (D, lanes 2, 4, 6). Total
RNA was isolated from leaves (lanes 1 and 2), stems (lanes 3 and
4), and roots (lanes 5 and 6) of either light-grown (L) or
dark-adapted (D) plants. Gene-specific probes from the 3'
non-coding region of AS1 and AS2 cDNAs were used to detect AS mRNA
(2.2 kb). As a control, cytosolic GS (1.4 kb) mRNA was also
monitored on the Northern blot (Tingey et al., 1987, EMBO J.
6:1-9). These results demonstrate that the dark-induced
accumulation of AS1 and AS2 mRNA occurs independent of
organ-type.
FIG. 8(parts a-c). Circadian rhythm is not involved in the
dark-induced expression of AS1 gene. Pea plants were grown under a
normal light/dark cycle (16 hours of light and 8 hours of dark) for
18 days. On the 19th day, some of the plants were kept in the
normal light/dark cycle (A), some of the plants were kept in
extended darkness for a total of 18 hours (B), and others were kept
in extended light condition instead of darkness (C). Leaves were
collected every 3 hours on the 19th day and used for isolating
total RNA. Gene-specific Northern blot analyses were performed to
detect the steady-state levels of AS1 mRNA. As a control, mRNA
levels of cytosolic GS were detected in the same blots. Empty boxes
represent light period, and black boxes represent dark period.
FIG. 9. Dark-induced expression of AS genes is regulated at the
transcriptional level. Transciptional analysis of AS genes in pea
leaves was assayed in nuclear run-on experiments. Nuclei isolated
from leaves of dark-adapted (D) or light-grown (L) pea plants were
used to perform nuclear run-on assays with .sup.32 P-ATP (Hagen et
al., 1985, Mol. Cell. Biol. 5:1197-1203; Kuo et al., 1988, Mol.
Cell. Biol. 8:4966-4971). Slot-blot filters containing the AS1 and
AS2 cDNA clones, positive controls (cDNA clones of chloroplast GS
and cytosolic GS), and a negative control (pTZ18U) were used to
detect specific .sup.32 P-labeled transcripts generated in the
nuclear run-on reactions. These results demonstrate that the
dark-induced expression of AS genes is regulated at the
transcriptional level.
FIG. 10 (parts a-c). Nucleotide sequence of the AS1 promoter
numbered according to the transcription start site. The 5' upstream
sequence of AS1 gene is shown and nucleotide number 1 corresponds
to the transcription start site. The "TATA" element at nucleotide
number -30 is boxed.
FIG. 11. Deletion analysis of the AS1 promoter defines regions of
DNA required for expression in vivo. Different regions of the 5'
upstream sequence of the AS1 gene were subcloned into pBI101.1
plasmid in a transcriptional fusion with the GUS reporter gene
(Jefferson et al., 1987, EMBO J. 6:3901-3907). AS-GUS constructs
were introduced into transgenic plants via Agrobacterium mediated
DNA transfer (Horsch et al., 1985, Science 227:1229-1231). gAS1a is
a genomic fragment containing DNA sequences of the AS1 gene from
nucleotide -2376 to +877 (+1 represents the transcription
initiation site). Regions of the AS1 promoter were fused to GUS as
follows: construct pBI-AS1001 contains nucleotides -2376 to +11 of
the AS1 promoter; construct pBI-AS1002 contains nucleotides -1373
to +11 of the AS1 promoter; and construct pBI-AS1003 contains
nucleotides -558 to +11 of the AS1 promoter. For construct
pBI-1004, an internal fragment from nucleotide -1373 to -558 of the
AS1 promoter was deleted. The ability of each AS promoter fragment
to direct expression of the GUS reporter gene was monitored by GUS
activity assay performed on leaves of dark-adapted transgenic
tobacco (Jefferson et al., 1987, Plant. Mol. Biol. Reporter
5:387-405). "+" represents the constructs in which GUS activity was
detected in transgenic plants. The GUS activity for pBI-AS1002 and
pBI-AS1004 have not yet been determined (N.D.).
FIG. 12(parts a and b). Nucleotide sequence of the AS2 promoter.
The 5' upstream sequence of the AS2 gene was determined by
sequencing serial deletion clones of the gAS2a BamHI-BamHI genomic
insert. Nucleotide number 1 represents the end of one deletion
clone used for sequencing. The putative "TATA" element at
nucleotide 989 is boxed, and the 5' end of AS2 cDNA is marked by an
arrow.
FIG. 13. The AS1 and AS2 promoters contain conserved sequence
elements which may be involved in the regulation of transcription.
Conserved DNA sequences between AS1 and AS2 promoters are shown.
The 569 nucleotides of the AS1 promoter (-558 to +11) were compared
to the entire AS2 promoter sequence shown in FIG. 12. Conserved
sequences shown in FIG. 13 were detected using computer program
DNASIS.
5. DESCRIPTION OF THE INVENTION
The present invention relates to the cloning and expression of
biologically active plant AS, an important but poorly understood
enzyme involved in the formation of asparagine--one of the major
nitrogen transport compounds of higher plants. The invention also
relates to the plant AS promoter which directs high levels of
expression in nitrogen fixing root nodules, in cotyledons of
germinating seeds, and dark-induced gene expression in leaves,
stems and roots. This promoter can be used to direct the regulated
expression of heterologous gene sequences in appropriate hosts.
In accordance with one aspect of the invention, biologically active
plant AS may be produced by the cloning and expression of the
nucleotide coding sequence for plant AS or its functional
equivalent in appropriate host cells. Successful expression and
production of purified plant AS using the full length cDNA clones
described and exemplified herein is particularly significant since
this enzyme has yet to be purified to homogeneity from plants
despite years of intensive biochemical investigation due to a
number of factors present in the plant extracts. E.g., the extreme
instability of AS in partially purified plant extracts; the
presence of contaminating asparaginase activity in the plant
extracts making it difficult to assay for AS activity; and the
presence of specific AS inhibitors in the plant extracts.
The synthesis of purified plant AS enzyme via genetic engineering
techniques which provide for the expression of full length cDNAs
may be utilized to obtain antibodies specific for plant AS which
will enable the further characterization of the distinct forms and
subcellular localizations of AS enzymes in plant cells. In
particular, the subcellular localization of plant AS to the
plastids would be significant since a plastid isoform of plant AS
may have very different properties in terms of herbicide resistance
as compared to cytosolic isoforms of AS. Such studies, if attempted
with antibodies that define mammalian AS could not be used to
address these issues. The ability to distinguish plastid and
cytosolic isoforms of AS, and to identify herbicides that
specifically inhibit the plastid isoform, but not cytosolic AS or
human AS, is important for designing herbicides that are not toxic
or harmful to humans and animals. In general, herbicides in current
use are inhibitors of enzymes involved in the formation of
essential amino acids, i.e., enzymes and amino acids which are not
produced by animals. The ability to develop herbicides that inhibit
only chloroplast isoforms of enzymes such as AS or GS (i.e.,
enzymes involved in the formation of non-essential amino acids) but
do not inhibit cytosolic AS or human AS, would vastly expand the
repertoire of herbicides which could be used having greater
efficacy and less toxicity to humans.
The plant AS cDNA clones described herein also can be used to
engineer herbicide resistant plants, salt/drought tolerant plants,
and pathogen resistant plants, or to screen and select novel
herbicides or agents which synchronize cells in culture. For
example, AS cDNAs genetically altered in vitro or in vivo can be
used to produce altered AS enzymes which are resistant to known AS
inhibitors. Such genes can be engineered into transgenic plants to
confer herbicide resistance to the plant. Similarly, herbicide
resistance, salt tolerance or drought tolerance may be conferred by
engineering the over-expression of wild-type or altered AS in
transgenic plants so that asparagine is overproduced. Expression of
such wild type or altered AS can be used as a dominant selectable
marker in a variety of organisms. Alternatively, the cloned plant
AS cDNAs expressed by microorganisms can be used to screen and
identify new herbicidal compounds, or cell cycle inhibitors that
selectively inhibit the AS holoenzyme.
The invention may be divided into the following stages solely for
the purpose of description: (a) isolation or generation of the
coding sequence for plant AS gene(s); (b) construction of an
expression vector which will direct the expression of the plant AS
coding sequences; (c) transfection of appropriate hosts which are
capable of replicating and expressing the plant AS coding sequences
to produce biologically active gene products; and (d)
identification and/or purification of the plant AS so produced.
Once a transformant or transfectant is identified that expresses
high levels of biologically active plant AS, the practice of the
invention involves the expansion of that clone and the use of that
clone in the production of plant AS enzymes, the selection of novel
herbicides, and/or the engineering of transgenic plants.
Another aspect of the invention involves the use of the plant AS
promoter to direct the expression of heterologous coding sequences.
The AS promoter directs high levels of expression in nitrogen
fixing root nodules, in cotyledons of germinating seeds, and
dark-induced gene expression in leaves, stems and roots. This
promoter can be used to direct the regulated expression of
heterologous gene sequences in appropriate hosts.
The invention is demonstrated herein, by way of examples in which
cDNAs of plant AS were prepared, cloned and characterized. Sequence
analysis of the plant AS cDNA clones, which were isolated using
heterologous DNA probes encoding human AS, revealed two different
plant AS cDNAs that encode homologous but distinct plant AS
polypeptides, AS1 and AS2. The organ-specific and "photophobic"
expression of AS1 and AS2 mRNAs in higher plants in vivo is also
described. Genomic clones of AS containing the plant AS photophobic
promoter were also prepared, cloned and sequenced.
Various aspects of the invention are described in more detail in
the subsections below and in the examples that follow.
5.1. PLANT ASPARAGINE SYNTHETASE CODING SEQUENCE
The nucleotide coding sequences and deduced amino acid sequences
for plant AS1 and AS2 are depicted in FIG. 2 (FIG. 2A and 2B,
respectively). These nucleotide sequences, or fragments or
functional equivalents thereof, may be used to generate recombinant
DNA molecules that direct the expression of the AS1 or AS2 gene
product, or functionally active peptides or functional equivalents
thereof, in appropriate host cells.
Due to the degeneracy of the nucleotide coding sequences, other DNA
sequences which encode substantially the same amino acid sequences
as depicted in FIGS. 2A and 2B may be used in the practice of the
present invention for the cloning and expression of plant AS. Such
alterations include deletions, additions or substitutions of
different nucleotide residues resulting in a sequence that encodes
the same or a functionally equivalent gene product. The gene
product may contain deletions, additions or substitutions of amino
acid residues within the sequence, which result in a silent change
thus producing a bioactive product. Such amino acid substitutions
may be made on the basis of similarity in polarity, charge,
solubility, hydrophobicity, hydrophilicity and/or the amphipathic
nature of the residues involved. For example, negatively charged
amino acids include aspartic acid and glutamic acid; positively
charged amino acids include lysine and arginine; amino acids with
uncharged polar head groups having similar hydrophilicity values
include the following: leucine, isoleucine, valine; glycine,
alanine; asparagine, glutamine; serine, threonine; phenylalanine,
tyrosine.
The genomic sequences for plant AS may be obtained from any plant
cell source, whereas mRNA for preparation of cDNA copies may be
obtained from cell sources that produce AS. For example, parts of
plants (e.g., leaves, stems, roots, nodules, cotyledons, seeds,
fruits, etc.) may be ground and used as the source for extracting
DNA or RNA. Alternatively, plant cell lines can be used as a
convenient source of DNA or RNA. Genetically engineered
microorganisms or cell lines containing plant AS coding sequences,
such as the deposited embodiments described herein, may be used as
a convenient source of DNA for this purpose.
The AS coding sequence may be obtained by cDNA cloning of RNA
isolated and purified from such cellular sources or by genomic
cloning. Either cDNA or genomic libraries may be prepared from the
DNA fragments generated using techniques well known in the art,
including but not limited to the use of restriction enzymes. The
fragments which encode plant AS may be identified by screening such
libraries with a nucleotide probe that is substantially
complementary to any portion of the sequences depicted in FIG. 2.
Although portions of the coding sequence may be utilized, full
length clones, i.e, those containing the entire coding region for
AS, may be preferable for expression. To these ends, techniques
well known to those skilled in the art for the isolation of DNA,
generation of appropriate restriction fragments, construction of
clones and libraries, and screening recombinants may be used. For a
review of such techniques see, for example, Maniatis et al., 1982,
Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press,
N.Y., Chapters 1-11. Alternatively, oligonucleotides derived from
AS1 or AS2 sequences could be used as heterologous primers in PCR
(polymerase chain reactions) to generate cDNA or genomic copies of
AS sequences from other species. For a review of such PCR
techniques, see for example, Gelfand, D. H., 1989, "PCR Technology.
Principles and Applications for DNA Amplification," Ed., H. A.
Erlich, Stockton Press, N.Y.; and "Current-Protocols in Molecular
Biology," Vol. 2, Ch. 15, Eds. Ausubel et al., John Wiley &
Sons, 1988.
In an alternate embodiment of the invention, the coding sequence of
FIG. 2A or 2B could be synthesized in whole or in part, using
chemical methods well known in the art. See, for example,
Caruthers, et al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233; Crea
and Horn, 180, Nuc. Acids Res. 9(10): 2331; Matteucci and
Caruthers, 1980, Tetrahedron Letters 21:719; and Chow and Kempe,
1981, Nuc. Acids Res. 9(12) 2807-2817. Alternatively, the protein
itself could be produced using chemical methods to synthesize the
amino acid sequence depicted in FIG. 2A or FIG. 2B in whole or in
part. For example, peptides can be synthesized by solid phase
techniques, cleaved from the resin, and purified by preparative
high performance liquid chromatography. (E.g., see, Creighton,
1983, Proteins Structures And Molecular Principles, W. H. Freeman
and Co., N.Y. pp. 50-60). The composition of the synthetic peptides
may be confirmed by amino acid analysis or sequencing (e.g., the
Edman degradation procedure; see Creighton, 1983, Proteins,
Structures and Molecular Principles, W. H. Freeman and Co., N.Y.,
pp. 34-49).
In the specific embodiments described in the examples herein, the
plant AS coding sequence was obtained by direct cloning of AS cDNAs
from Pisum sativum using a heterologous DNA probe for human AS.
Nucleotide sequence analysis of these cDNAs demonstrate that peas
contain two homologous but distinct AS mRNAs, AS1 and AS2 (FIGS. 1
and 2). The two distinct cDNAs (pcAS1 and pcAS2) encode homologous
but distinct AS polypeptides. Homology between plant AS and human
AS was determined to be approximately 47%, demonstrating that human
and plant AS are evolutionarily related, yet quite distinct
molecules (FIG. 3, and Section 6.2.2 at Table II, infra). Southern
blot analysis of pea nuclear DNA revealed that AS is encoded by a
small gene family in peas consisting of at least two genes, AS1 and
AS2 (FIG. 4). Northern blot analysis of pea RNA revealed that the
AS1 and AS2 mRNAs accumulate differentially during plant
development (FIGS. 5 and 6).
There are numerous developmental contexts where high levels of
asparagine are synthesized for nitrogen transport. When plants are
grown in the dark, asparagine is the preferred nitrogen transport
compound compared to glutamine since it has a high N:C ratio,
making it a more economical compound for nitrogen transport and
storage under growth conditions where carbon skeletons are limited.
Consistent with these physiological findings, our analysis of AS
mRNA has shown that AS1 mRNA accumulates to high levels in leaves
of dark-grown or dark-adapted plants (FIG. 5) and in roots and
stems (FIG. 7). AS1 mRNA accumulates to high levels within as
little as 3 hours of darkness. Thus the "photophobic" accumulation
of AS1 mRNA is physiologically significant for plants grown in a
short dark cycle (i.e., at night). In contrast, AS2 mRNA is present
at low levels in leaves of light or dark grown plants.
Asparagine is also the preferred nitrogen transport/storage
compound in two other developmental contexts where large amounts of
nitrogen must be mobilized, namely in the transport of nitrogen out
of cotyledons to the seedling during germination, and the transport
of newly fixed nitrogen out of root nodules to the rest of the
plant. In the majority of seeds, nitrogen is stored in the
cotyledons as insoluble proteins called seed storage proteins.
During germination, these seed storage proteins provide a ready
reserve of nitrogen for the growing seedling. The amino acids
produced by hydrolysis of the storage proteins in the cotyledons
are rarely transported out of the cotyledons as such. The released
amino acids are first deaminated, and the majority of the ammonia
is transported out of the cotyledons as either glutamine or
asparagine. A classic example of asparagine synthesis occurs in
lupin where 86.5% of the nitrogen from seed storage protein is
converted into asparagine. Asparagine is also the major amino acid
transported out of cotyledons in germinating cotton seedlings,
wheat grains (Garg et al 1984, J. Agricult. Food Chem. 32
(3):519-523 , soybean, maize (Oaks & Ross, 1984, Can J. Bot. 62
(10): 68-73), and apple seeds. For review, see Lea & Miflin,
1980, Transport And Metabolism Of Asparagine And Other Nitrogen
Compounds Within the Plant, in The Biochemistry Of Plants, Vol. 5,
Ed. Miflin, Acad. Press, N.Y., Ch. 16.
We have shown that the pivotal role of asparagine in cotyledons of
germinating peas is reflected at the molecular level. mRNAs for
both AS1 and AS2 accumulate to high levels in cotyledons of
germinating pea seedlings (FIG. 6A). In amide-transporting legumes
(e.g., peas, alfalfa) the majority of newly fixed ammonia is
transported out of nitrogen-fixing root nodules as glutamine or
asparagine. AS shows the most dramatic increase in activity when
the enzymes of N-metabolism are examined in alfalfa root nodules.
The role of AS in asparagine synthesis in nodules is also reflected
at the molecular level. As demonstrated herein, AS1 and AS2 mRNAs
accumulate to high levels in nitrogen fixing root nodules of pea,
where newly fixed ammonia must be transported from the root nodules
to the developing plant (FIG. 6B).
5.2. EXPRESSION OF PLANT ASPARAGINE SYNTHETASE
In order to express a biologically active plant AS, the nucleotide
sequence coding for plant AS, or a functional equivalent as
described in Section 5.1 supra, is inserted into an appropriate
expression vector, i.e., a vector which contains the necessary
elements for the transcription and translation of the inserted
coding sequence. The plant AS gene product as well as host cells,
cell lines or plants transfected or transformed with recombinant AS
expression vectors can be used for a variety of purposes. These
include but are not limited to generating antibodies (i.e.
monoclonal or polyclonal) that define plant AS; creating mutant AS
enzymes which are resistant to herbicides; creating transgenic
plants containing such herbicide resistant mutant AS genes; or
creating transgenic plants which over-express AS and demonstrate
herbicide resistance, salt/drought tolerance and/or pathogen
resistance; screening and selecting for novel AS inhibitors which
may be used as herbicides, and/or cell cycle regulators for
synchronizing plant cells in culture.
5.2.1. CONSTRUCTION OF EXPRESSION VECTORS CONTAINING THE PLANT AS
CODING SEQUENCE
Methods which are well known to those skilled in the art can be
used to construct expression vectors containing the AS coding
sequence and appropriate transcriptional/translational control
signals. These methods include in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Maniatis et al., 1982 Molecular Cloning A Laboratory Manual, Cold
Spring Harbor Laboratory, N.Y., Chapter 12.
A variety of host-expression vector systems may be utilized to
express the plant AS coding sequence. These include but are not
limited to microorganisms such as bacteria transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing the plant AS coding sequence; yeast transformed
with recombinant yeast expression vectors containing the plant AS
coding sequence; insect cell systems infected with recombinant
virus expression vectors (e.g., baculovirus) containing the plant
AS coding sequence; plant cell systems infected with recombinant
virus expression vectors (e.g., cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing the plant AS
coding sequence; or animal cell systems infected with recombinant
virus expression vectors (e.g., adenovirus, vaccinia virus)
containing the plant AS coding sequence.
The expression elements of these vectors vary in their strength and
specificities. Depending on the host/vector system utilized, any of
a number of suitable transcription and translation elements,
including constitutive and inducible promoters, may be used in the
expression vector. For example, when cloning in bacterial systems,
inducible promoters such as pL of bacteriophage .lambda., plac,
ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used;
when cloning in insect cell systems, promoters such as the
baculovirus polyhedrin promoter may be used; when cloning in plant
cell systems, promoters derived from the genome of plant cells
(e.g., heat shock promoters; the promoter for the small subunit of
RUBISCO; the promoter for the chlorophyll a/b binding protein) or
from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat
protein promoter of TMV) may be used; when cloning in mammalian
cell systems, promoters derived from the genome of mammalian cells
(e.g., metallothionein promoter) or from mammalian viruses (e.g.,
the adenovirus late promoter; the vaccinia virus 7.5K promoter) may
be used. Promoters produced by recombinant DNA or synthetic
techniques may also be used to provide for transcription of the
inserted plant AS coding sequence.
In bacterial systems a number of expression vectors may be
advantageously selected depending upon the use intended for the
plant AS expressed. For example, when large quantities of AS are to
be produced for the generation of AS antibodies, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include
but are not limited to the E. coli expression vector pUR278 (Ruther
et al., 1983, EMBO J. 2:1791), in which the plant AS coding
sequence may be ligated into the vector in frame with the lac Z
coding region so that a hybrid AS-lac Z protein is produced; pIN
vectors (Inouye & Inouye, 1985, Nucleic acids Res.
13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
264:5503-5509); and the like. However, where the plant AS
expression vector is to be used in an Asn.sup.- host for
complementation assays described infra, the expression of unfused
AS using expression vectors with few or no host genotype
requirements, including, but not limited to vectors such as ptac12,
(Amann et al., 1983, Gene 25:167) and the like may be
preferred.
In yeast, a number of vectors containing constitutive or inducible
promoters may be used. For a review see, Current Protocols in
Molecular Biology, Vol. 2, 1988, Ed. Ausubel et al., Greene
Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al.,
1987, Expression and Secretion Vectors for Yeast, in Methods in
Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol.
153, pp.516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press,
Washington, D.C., Ch. 3; and Bitter, 1987, Heterologous Gene
Expression in Yeast, Methods in Enzymology, Eds. Berger &
Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular
Biology of the Yeast Saccharomyces, 1982, Eds. Strathern et al.,
Cold Spring Harbor Press, Vols. I and II. For complementation
assays in yeast, pea cDNAs for AS may be cloned into yeast episomal
plasmids (YEp) which replicate autonomously in yeast due to the
presence of the yeast 2.mu. circle. The plant AS sequence may be
cloned behind either a constitutive yeast promoter such as ADH or
LEU2 or an inducible promoter such as GAL (Cloning in Yeast, Chpt.
3, R. Rothstein In: DNA Cloning Vol.11, A Practical Approach, Ed.
DM Glover, 1986, IRL Press, Eash., D.C.). Constructs may contain
the 5' and 3' non-translated regions of the cognate plant mRNA or
those corresponding to a yeast gene. YEp plasmids transform at high
efficiency and the plasmids are extremely stable. Alternatively
vectors may be used which promote integration of foreign DNA
sequences into the yeast chromosome.
In cases where plant expression vectors are used, the expression of
the plant AS coding sequence may be driven by any of a number of
promoters. For example, viral promoters such as the 35S RNA and 19S
RNA promoters of CaMV (Brisson et al., I984, Nature 310:511-514),
or the coat protein promoter of TMV (Takamatsu et al., 1987, EMBO
J. 6:307-311) may be used; alternatively, plant promoters such as
the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.
3:1671-1680; Broglie et al., 1984, Science 224:838-843); or heat
shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et
al., 1986, Mol. Cell. Biol. 6:559-565) may be used. These
constructs can be introduced into plant cells using Ti plasmids, Ri
plasmids, plant virus vectors, direct DNA transformation,
microinjection, electroporation, etc. For reviews of such
techniques see, for example, Weissbach & Weissbach, 1988,
Methods for Plant Molecular Biology, Academic Press, NY, Section
VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular
Biology, 2d Ed., Blackie, London, Ch. 7-9.
An alternative expression system which could be used to express
plant AS is an insect system. In one such system, Autographa
californica nuclear polyhedrosis virus (AcNPV) is used as a vector
to express foreign genes. The virus grows in Spodoptera frugiperda
cells. The plant AS coding sequence may be cloned into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter). Successful insertion of the plant AS
coding sequence will result in inactivation of the polyhedrin gene
and production of non-occluded recombinant virus (i.e., virus
lacking the proteinaceous coat coded for by the polyhedrin gene).
These recombinant viruses are then used to infect Spodoptera
frugiperda cells in which the inserted gene is expressed. (E.g.,
see Smith et al., 1983, J. Viol. 46:584; Smith, U.S. Pat. No.
4,215,051).
In cases where an adenovirus is used as an expression vector, the
plant AS coding sequence may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene may then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a nonessential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing plant AS in infected
hosts. (E.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci.
(USA) 81:3655-3659). Alternatively, the vaccinia 7.5K promoter may
be used. (E.g., see Mackett et al., 1982, Proc. Natl. Acad. Sci.
(USA) 79:7415-7419; Mackett et al., 1984, J. Virol. 49:857-864;
Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931).
Specific initiation signals may also be required for efficient
translation of inserted AS coding sequences. These signals include
the ATG initiation codon and adjacent sequences. In cases where the
entire AS gene, including its own initiation codon and adjacent
sequences, are inserted into the appropriate expression vectors, no
additional translational control signals may be needed. However, in
cases where only a portion of the AS coding sequence is inserted,
exogenous translational control signals, including the ATG
initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the AS coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bitter et al., 1987, Methods in Enzymol.
153:516-544).
In addition, a host cell strain may be chosen which modulates the
expression of the inserted sequences, or modifies and processes the
gene product in the specific fashion desired. Expression driven by
certain promoters can be elevated in the presence of certain
inducers, (e.g., zinc and cadmium ions for metallothionein
promoters). Therefore, expression of the genetically engineered
plant AS may be controlled. This is important if the protein
product of the cloned foreign gene is lethal to host cells.
Furthermore, modifications (e.g., glycosylation) and processing
(e.g., cleavage) of protein products may be important for the
function of the protein. Different host cells have characteristic
and specific mechanisms for the post-translational processing and
modification of proteins. Appropriate cells lines or host systems
can be chosen to ensure the correct modification and processing of
the foreign protein expressed.
One way to select for the expression of a biologically active plant
AS gene product, be it wild type, mutated or altered, and/or to
screen for new herbicides, is to introduce an appropriate
expression vector containing the AS coding sequence into asparagine
minus (Asn ) host cell strains (of bacteria, algae, neurospora,
yeast, higher plants or animals) which are grown in selective
asparagine minus media, as in the complementation assay described
infra. The ability of an AS cDNA to confer asparagine independent
growth to the host cells, i.e., complement the host cells,
indicates the expression and formation of an active holoenzyme. A
number of different (Asn.sup.-) host cell strains may be used for
this purpose including but not limited to asnA.sup.-, asnB.sup.-
strains of E. coli, JE6279 (Nakamura, et al., 1981, Nuc. Acids Res.
9:4669-4676) or ER (Kaguni et al., 1979 Proc. Natl. Acad. Sci.
(USA) 76:6250-6254): neurospora Asn hosts (MacPhee et al., 1983, J.
Bacteriol. 156:457-478); asn1.sup.-, asn2.sup.- yeast host cells
such as XE299-1A XE293-8B, or XE293-8D (Jones et al., 1978, J.
Bacteriol. 134:200-207); and plant cell lines such as the gln.sup.-
asn.sup.- soybean cell line (Roth & Lark, 1982, Plant Cell
Reports 1:157-160), to name but a few. Heterologous host systems
may require the use of a second selectable marker as a means for
selecting incorporation of the vector, e.g., in cases where plant
AS may not complement the Asn.sup.- auxotroph. To this end, rescue
of auxotrophy for another amino acid in addition to Asn (e.g.,
histidine) can be used as the selectable marker; alternatively,
dominant selectable markers such as dhfr and resistance to
methotrexate, thymidine kinase activity, etc. described infra. may
be used. For example, the yeast YEp plasmids have been constructed
to contain a number of different selectable marker genes such as:
his3, ura3, leu2 or trpl (Ma et al., 1987, Gene 58: 201-216). Yeast
strains harboring the auxotrophy compatible with one of these YEp
vectors may be crossed into an asn.sup.- strain, e.g., XE293-8B or
8D (Jones, 1978, J. Bacteriol. 134:200-207) using standard yeast
genetic techniques. The yeast strain harboring the asn.sup.-
phenotype and marker auxotrophy is transformed with the YEp vector
containing plant AS; asn.sup.+ colonies may then be selected and
characterized.
5.2.2. IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS EXPRESSING
THE PLANT AS GENE PRODUCT AND ISOLATION OF PLANT AS
The host cells which contain the plant AS coding sequence and which
express the biologically active AS gene product may be identified
by at least four general approaches: (a) DNA-DNA hybridization; (b)
the presence or absence of "marker" gene functions; (c) assessing
the level of transcription as measured by the expression of AS mRNA
transcripts in the host cell; and (d) detection of the AS gene
product as measured by immunoassay or by its biological activity;
(e) phenotypic rescue; or (f) resistance to herbicides.
In the first approach, the presence of the plant AS coding sequence
inserted in the expression vector can be detected by DNA-DNA
hybridization using probes comprising nucleotide sequences that are
homologous to the plant AS coding sequence substantially as shown
in FIG. 2A or 2B or portions or derivatives thereof.
In the second approach, the recombinant expression vector/host
system can be identified and selected based upon the presence or
absence of certain "marker" gene functions (e.g., thymidine kinase
activity, resistance to antibiotics, resistance to methotrexate,
transformation phenotype, occlusion body formation in baculovirus,
etc.). For example, if the plant AS coding sequence in inserted
within a marker gene sequence of the vector, recombinants
containing the plant AS coding sequence can be identified by the
absence of the marker gene function. Alternatively, a marker gene
can be placed in tandem with the plant AS sequence under the
control of the same or different promoter used to control the
expression of the plant AS coding sequence. Expression of the
marker in response to induction or selection indicates expression
of the plant AS coding sequence. Two marker gene constructs which
may be of particular value for monitoring promoter activity in
plant cells and plants are the bacterial glucuronidase gene, GUS
(Jefferson et al., 1987, EMBO J. 6:3901-3908) or the luciferase
gene (Ow et al., 1987, Science, 234:856-859)
In the third approach, transcriptional activity for the plant AS
coding region can be assessed by hybridization assays. For example,
RNA can be isolated and analyzed by Northern blot using a probe
homologous to the plant AS coding sequence or particular portions
thereof substantially as shown in FIG. 2. Alternatively, total
nucleic acids of the host cell may be extracted and assayed for
hybridization to such probes.
In the fourth approach, the expression of the AS protein product
can be assessed immunologically, for example by Western blots,
immunoassays such as radioimmunoprecipitation, enzyme-linked
immunoassays and the like. The ultimate test of the success of the
expression system, however, involves the detection of the
biologically active plant AS gene product. Where the host cell
secretes the gene product, the cell free media obtained from the
cultured transfectant host cell may be assayed for plant AS
activity. Where the gene product is not secreted, cell lysates may
be assayed for such activity. In either case, a number of assays
can be used to detect AS activity including but not limited to
measuring the conversion of .sup.14 C-aspartate to .sup.14
C-asparagine (Joy et al., 1983, Plant Physiol. 73:165-168, Shelp
& Atkins, 1984, Plant Science Letters 36:225-230; Huber &
Streeter, 1984, Plant Physiol. 74:605-610; Huber et al., 1985,
Plant Sci. 42:9-17); measuring such conversion by absorption
(Rognes, 1975, Phytochem. 14:1975-1982; Loyola-Vargas, 1988, J.
Plant Physiol. 132:289-293); or measuring AS by HPLC (Unnithan et
al., 1984, Anal. Biochem. 136:198-201).
In the fifth approach, the production of a biologically active
plant AS gene product can be assessed by the complementation assay,
in which an Asn.sup.- host transformed or transfected with the AS
expression vector is grown on asparagine minus media. The
expression of a biologically active holoenzyme is indicated by the
growth of such transformants/transfectants in the absence of
asparagine. As previously explained, this may be used in
conjunction with a separate selectable marker to ensure
incorporation of the vector.
In the sixth approach, mutant or wild-type plant AS which is
resistant to herbicides can be selected using the complementation
assay expression system by exposing the clones which express
biologically active AS to various concentrations of different
herbicides. Growth in an asparagine minus media in the presence of
the herbicide indicates expression of a resistant, active
holoenzyme.
Once a clone that produces high levels of biologically active plant
AS is identified, the clone may be expanded and used for a variety
of ends; e.g., production of plant AS which may be purified using
techniques well known in the art including but not limited to
immunoaffinity purification, chromatographic methods including high
performance liquid chromotography, and the like; screening
herbicides; and engineering transgenic plants which are herbicide
resistant, salt/drought tolerant and/or pathogen resistant.
5.3. USES OF PLANT ASPARAGINE SYNTHETASE GENE AND GENE PRODUCT
Our studies concerning AS mRNA accumulation have highlighted the
importance of the two different AS mRNAs during plant development.
The AS cDNA clones can be used to characterize the distinct AS1 and
AS2 gene products; to express AS so that antibodies which define
AS1 or AS2 gene products can be produced; to screen and develop new
herbicides; to develop herbicide resistant plants, salt/drought
tolerant plants or pathogen resistant plants; and to aid in the
identification of novel cell cycle inhibitors which can be used to
synchronize plant cell cultures.
5.3.1. PRODUCTION OF ANTIBODIES THAT DEFINE AND/OR INHIBIT PLANT
AS
Expressed gene products may be used to produce antibodies that
define AS1 or AS2. These antibodies can be used in organelle
fractionation studies to define the subcellular site of action of
these distinct AS polypeptides so that previous biochemical studies
which report either cytosolic or plastid localization of AS
activity in plant cells can be clarified. For full length cDNA
clones, the corresponding proteins may be produced in E. coli in
sufficient quantities to allow characterization in terms of
substrate preference (i.e. ammonia or glutamine), Km, sensitivity
to inhibitors, etc.
Such antibodies may be produced by any method known in the art,
including, but not limited to injection of plant AS into mice,
rats, rabbits, or other host species for production of polyclonal
antisera. Various adjuvants may be used to increase the immune
response. These include, but are not limited to Freund's (complete
and incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, and oil emulsions. For a review of such
techniques, see Harlow & Lane, 1988, Antibodies A Laboratory
Manual, Cold Spring Harbor Laboratory.
Monoclonal antibodies, or fragments thereof, can be prepared by any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. Such techniques include but
are not limited to the hybridoma technique first developed by
Kohler and Millstein (1975, Nature 256:495-497), the human B-cell
hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72),
and the EBV-hybridoma technique for production of human monoclonal
antibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96).
Antibody fragments which contain the idiotype of the molecule could
be generated by known techniques. For example, such fragments
include but are not limited to: the F(ab').sub.2 fragment which can
be produced by pepsin digestion of the antibody molecule; the Fab'
fragments which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragment, and the 2 Fab or Fab fragments which
can be generated by treating the antibody molecule with pepsin and
a reducing agent.
5.3.2. DEVELOPMENT OF NEW HERBICIDES
AS cDNA clones can be used in functional complementation assays
described in Section 5.1, supra, designed to screen for new
herbicides as follows. Full length AS cDNAs cloned in the
appropriate expression vectors can be introduced into Asn strains
of bacteria, algae yeast, neurospora, or higher plant or animal
cells as previously described. The ability of an AS cDNA clone to
confer asparagine independent growth to the Asn.sup.- mutant cells
would indicate that the cDNA encodes an AS polypeptide which is
able to assemble into an active holoenzyme in these heterologous
environments.
The in vivo complementation experiments will determine whether AS1
and AS2 encode separate subunits of separate holoenzyme complexes
or whether they encode two different subunits of a single
holoenzyme complex. That a plant enzyme for AS can assemble and
function in a heterologous environment is supported by the findings
that cDNAs for higher plant GS can functionally rescue GlnA.sup.-
strains of E. coli even though the plant and E. coli enzymes differ
dramatically (Das Sarma, 1986, Science, 232:1242-1244; Snustad et
al., 1988, Genetics Soc. America vol. 120 (4):1112-1124).
The plant AS cDNAs expressed in Asn.sup.- mutants of bacteria,
algae, yeast, neurospora, plant cells or animal cells described
previously provide an in vivo system to select for novel herbicides
which selectively act on AS. To this end, known concentrations of a
test substance can be added to the growth media in order to select
those substances which inhibit cell growth as an indication of AS
inhibitory activity.
5.3.3. DEVELOPMENT OF HERBICIDE RESISTANT PLANTS AND STRESS
TOLERANT PLANTS
The in vivo expression system described above could be modified and
used to select for mutations in the plant AS structural gene which
confer growth resistance to known herbicides or to the new
herbicides. Mutations may be introduced into the plant AS cDNA in
vivo or in vitro using techniques well known in the art, including,
but not limited to, radiation, chemical mutation, site-specific
mutations, etc. For example, see Tilghman and Levine, 1987, in Gene
Transfer, Kucherlaputi, Ed., Plenum Pub., N.Y., pp. 189-221).
Expression vectors containing such altered or mutated plant AS
coding sequences can be used in Asn.sup.- hosts in the
complementation assay described above to identify clones containing
coding sequences that specify and express biologically active,
altered plant AS. Clones which produce herbicide resistant plant AS
can be selected by growth in the presence of the herbicide. In this
way, clones which produce mutant plant AS can be screened for
resistance to various herbicides. Some known inhibitors of plant AS
which can be tested in this screening system are listed in Table I
below.
TABLE I ______________________________________ INHIBITORS OF PLANT
AS Inhibition (%) ______________________________________ Aspartate
Analogues erythro .beta.-hydroxy-L-aspartate 65.4 threo
.beta.-methyl-L-aspartate 58.4 erythro .beta.-methyl-L-aspartate
51.4 meso-diaminosuccinamate 50.6 5-bromo-4-oxo-L-norvaline 49.5
5-chloro-4-oxo-L-norvaline 42.1 Glutamine Analogues L-azaserine
97.5 L-albizziine 85.2 DL-homoglutamine 42.4 S-carbamoyl-L-cysteine
35.6 2-hydroxyethyl-L-glutamine 31.1 N-CBZ-DL-glutamine 30.5
L-glutamate-.gamma.-methyl ester 27.2 L-glutamate-.gamma.-ethyl
ester 23.6 .gamma.-methylene-L-glutamine 20.4 D-glutamine 14.8
N-acetyl-L-glutamine 10.2 L-glutamate diamide 9.1
L-methionine-S-sulphoximine 8.6 Asparagine Analogues
erythro-.beta.-hydroxy-L-asparagine 59.5 N-methyl-L-asparagine 57.6
.beta.-methyl-DL-asparagine 55.1 N-ethyl-L-asparagine 47.2 threo
.beta.-hydroxy-L-asparagine 45.2 2-amino-2-carboxy-L- 42.5
ethanesulphonamide 5-di azo-4-oxo-L-norvaline 40.3
N-.alpha.-methyl-L-asparagine 38.8
______________________________________ From Lea & Fowden, 1975,
Proc. R. Soc. London B. 192:13-26. Aspartate, glutamine or
asparagine analogues were added to the standard .sup.14 Caspartate
conversion assay mixtures at 2 mM, 5 mM and 2 mM, respectively The
reaction rate in the absence of any analogue was taken as zero
inhibition.
Plants may then be engineered for resistance to herbicides using
appropriate constructs containing the mutated AS genes which encode
resistant AS products as defined above. Alternatively, plants may
be engineered for resistance to herbicides by the over-expression
of wild-type AS. In either case, such transgenic plants may be
constructed using methods well known to those skilled in the art
including but not limited to techniques involving the use of the Ti
plasmids (e.g., Agrobacterium rhizogenes), plant viruses,
electroporation, direct transformation, microinjection, etc. By way
of example, and not by way of limitation, the binary Ti vector
system could readily be used to this end (Bevan, 1984, Nuc. Acids
Res. 12:8711-8721). This vector contains a selectable marker,
neomycin phosphotransferase (kanR), under direction of the nopaline
synthetase promoter (nos) for KanR selection in plants, and unique
cloning sites for EcoRI, HindIII, BamHI, Sma, Sal, Kpn, Kba, SstI.
Using this system, DNA fragments cloned into one of the unique
cloning sites located between the left and right T-DNA borders are
transferred into dicot plants when introduced into Agrobacterium
tumefaciens LB4404 harboring a resident diarmed Ti plasmid which
will provide vir gene required for T-DNA transfer in trans. Another
vector system which could be used is the pMON505 intermediate
binary Ti transformation vector (Horsch & Klee, 1986, Proc.
Natl. Acad. Sci. 83, 4428-4432; Horsch et al., 1985 Science,
227:1229-1231). For reviews of such techniques see, for example,
Weissbach & Weissbach, 1988, Methods for Plant Molecular
Biology, Academic Press, N.Y., Section VIII, pp. 421-463; and
Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed.,
Blackie, London, Ch. 7-9. AS genes altered in vivo or in vitro to
overproduce wild type AS or herbicide resistant forms of AS enzymes
may be used as dominant selectable markers for transformation
systems which include organisms such as bacteria, algae, yeast,
neurospora, plants and animal cells.
By way of illustration, herbicide resistant plants can be
engineered in at least two ways: AS cDNAs which contain mutations
conferring herbicide resistance as measured in the screening assay
above, can be introduced into transgenic plants under the
transcriptional regulation of a strong constitutive promoter (e.g.,
35S promoter of CaMV) or an inducible promoter (e.g., the promoter
of the small subunit of RUBISCO; a heat shock promoter, etc.).
Mutant AS produced in the transgenic plants will assemble with
wild-type subunits to confer resistance. Alternatively, herbicide
resistant plants could be developed by the over-expression of the
wild-type AS cDNA. The over-expression of AS in these transgenic
plants will produce plants resistant to high levels of a herbicide
specific for AS. While over-expression of other amino acid
biosynthetic genes may be detrimental to plant metabolism, the
over-expression of AS will not harm plant metabolism since
asparagine is an ideal nitrogen storage compound, and may in fact
actually benefit the plants in ways other than herbicide
resistance.
The overexpression of wild-type or mutant AS in transgenic plants
may confer resistance or tolerance to salt, drought and/or
pathogens. Asparagine has been shown to accumulate in a variety of
conditions in plants in response to stress. For example, asparagine
and proline accumulate in leaves of water-stressed plants and are
thought to serve to help maintain osmotic conditions. Free
asparagine has also been shown to accumulate in plants under growth
conditions of sulfur starvation (Baudet et al., 1986, Physiol.
Plant 68(4):608-614); increased salinity (Chen et al., 1988, Plant
Physiol. 86 (4 Suppl.):56 (Abstr); Jeschke et al., 1986, J. Plant
Physiol. 124: 257-274); drought (Drossopoulos et al., 1985, Ann.
Bot. London 56: 291-306; Fukutoku & Yamada, 1984, Physiol.
Plant 61:622-268) and also in plants infected with plant pathogens
(Walters & Ayres, 1980, Physiol. Plant Pathol. 17:369-380). The
over-production of asparagine in these contexts may endow the
transgenic plants with a protective mechanisms that enables them to
grow better than wild type. It has recently been shown in alfalfa
plants coinfected with the pathogen P. syringae and the symbiont R.
meliloti, that although GS activity is severely inhibited by toxin
(Tabtoxinine .beta.-lactam) produced by P. syringae, the infected
alfalfa plants assimilate greater total amounts of nitrogen than
their counterpart controls. This finding suggests that alternate
routes of ammonia assimilation (i.e., asparagine synthetase) can
occur in plants which confer better growth properties to a plant
(Knight & Langston-Unkefer, 1988, Science 241:951-952).
5.3.4. IDENTIFICATION OF AGENTS THAT SYNCHRONIZE PLANT CELLS IN
CULTURE
The screening assay described above could also be used to identify
AS inhibitors that could be tested as cell cycle inhibitors useful
for synchronizing plant cells in culture. It has been shown in
yeast that mutations in amino acid biosynthesis enzymes demonstrate
cell cycle defects (Wolfner et al., 1975, J. Mol. Biol.
96:273-290). It has been shown that cell cycle mutants in mammals
are deficient in asparagine synthetase (Greco et al., 1987, Proc.
Natl. Acad. Sci. 84:1565-1569). By analogy, inhibitors of AS
defined by the foregoing screening system could be tested as cell
cycle inhibitors to arrest growth of cells in culture, and
therefore, synchronize cells in culture. This may have wide
applicability to the production of man compounds in cell culture in
industry.
In another embodiment, AS genes altered in vivo or in vitro to
encode altered forms of AS could be introduced into a cell line
under transcriptional direction of an inducible promoter. Induced
expression of altered AS would arrest cells within the cell cycle
and act to synchronize the cell culture.
5.4. THE PLANT ASPARAGINE SYNTHETASE PROMOTER
As described above and exemplified below, the AS promoter directs
high levels of expression in nitrogen fixing root nodules in
cotyledons of germinating seeds, and dark-induced gene expression
in leaves. This promoter can be used to direct the regulated
expression of heterologous gene sequences in appropriate hosts.
Another promoter which exhibits photophobic transcription is the
promoter of the rice phytochrome gene (Hersey et al., 1985, Nuc.
Acids Res. 13:8543-8559; Hershey et al., 1987, Gene 61:339-348; Kay
et of transcription are not achieved with the phytochrome promoter.
By contrast, the plant AS promoter described herein is not only
inducible by darkness, but drives high levels of transcription.
The nucleotide sequence of the plant AS1 promoter is shown in FIG.
10, and the nucleotide sequence for the plant AS2 promoter is shown
in FIG. 12. Conserved DNA sequences of the plant AS1 and AS2
promoters are shown in FIG. 13.
The organ-specific and photophobic promoters can be used in a
variety of DNA vectors to drive the expression of heterologous
sequences ligated after the transcription start site indicated by
an arrow in FIG. 10 and upstream from the arrow shown in FIG. 12.
Heterologous sequences ligated downstream of the transcription
start site, containing a portion of the leader sequence, will
require translational control elements such as the ATG start
signal, and ribosome binding sites etc. Optionally, the
heterologous sequences may also be ligated in a translational
fusion to marker genes such .beta.-galactosidase, GUS, luciferase,
etc. to produce fusion proteins.
Such expression vectors may be used in plant cell culture or in
transgenic plants to direct high level expression of the
heterologous sequence in an inducible, temporal, or organ-specific
fashion.
As demonstrated by the examples described infra, isolated AS1
promoter was able to direct the expression of a GUS reporter gene
in leaves of dark-grown transgenic tobacco plants (see FIG. 11).
Results of in situ GUS stained leaves reveals that the AS1 promoter
directs GUS expression specifically in the phloem cells. Deletion
analysis revealed that the DNA sequences important for AS1 promoter
function are contained within a 569 bp fragment of the AS1 promoter
(see FIG. 11).
The AS2 promoter shares nucleotide homology with AS1 within the
region of the AS1 promoter required for gene expression. These
conserved DNA sequence elements (FIG. 13) may correspond to
cis-acting DNA elements responsible for AS1 and AS2 gene
expression.
6. EXAMPLE: cDNA CLONING OF PLANT ASPARAGINE SYNTHETASE
The following subsections describe the genomic cloning and cDNA
cloning of plant AS from pea cDNA and genomic libraries. Briefly,
human AS cDNA was used to screen a pea nodule cDNA library in order
to isolate AS1 cDNA clones by heterologous hybridization. Coding
regions of the AS1 cDNA were then utilized to screen a pea genomic
library, resulting in the isolation of AS1 and AS2 genomic clones.
The AS2 genomic clones were then utilized to screen a pea root cDNA
library in order to isolate AS2 cDNA clones.
6.1. MATERIALS AND METHODS
Seeds of P. sativum (var. "Sparkle") obtained from Rogers Brother
Seed Co. (Twin Falls, Id.) were imbibed and germinated in a
Conviron environmental chamber with a day length of 16 hours,
illumination of 1000 microeinsteins/m.sup.2 /s [1 einstein (E)=1
mol of photons], at a day/night cycle of 21.degree./18.degree. C.
For etiolated plants, peas were grown for 7-9 days in black lucite
boxes contained in a dark environmental chamber. For germination
studies, seeds were imbibed in water and germinated in vermiculite.
Nodules were isolated from 21 day old pea plants inoculated with
Rhizobium leguminosum strain 128C53 (Nitragin Co., Milwaukee, Wis.)
as described previously (Tingey et all, 1987, EMBO J. 6:1-9).
For phytochrome induction experiments, 9 day old etiolated pea
seedlings were irradiated with a 4 minute pulse of red light (red
fluorescent lamps, General Electric F20T12R) at a fluence of 40
.mu.E/m.sup.2 /s or were given a 4 minute pulse of red light
followed by 12 minutes of far-red light (Westlake, FRF700) at the
same fluence and were then returned to the dark for 3 hours. For
white light treatment, etiolated seedlings were exposed to
continuous white light for 8 hours.
The subsections below describe the methods used to isolate and
prepare the AS genomic and cDNA clones from the pea libraries. The
pea cDNA libraries used are described in Tingey et al, 1987, EMBO
J. 6:1-9, which is incorporated by reference herein in its
entirety.
6.1.1. ISOLATION OF PLANT AS1 cDNAs
AS cDNA clones were selected from a pea nodule cDNA library from
Pisum sativum (var. "Sparkle") in .lambda.gt11 (Tingey et al.,
1987, EMBO J. 6:1-9) as follows. Nitrocellulose filters containing
denatured phage DNA corresponding to 250,000 individual plaques
were incubated for 4 hours at 45.degree. C. in prehybridization
buffer (6.times.SSC 10.times.Denhardt's Solution, 0.1% SDS, 1 mM
EDTA, 100 .mu.g/ml denatured salmon sperm DNA). Filters were then
incubated for 24 hours at 45.degree. C. in hybridization buffer
(6.times.SSC, 5.times.Denhardt's Solution, 0.1% SDS, 1 mM EDTA, 50
.mu.g/ml denatured salmon sperm DNA) plus 0.2 .mu.g of .sup.32 P
labeled cDNA insert (a 1.7 kb Hind III fragment) of pH131 (Andrulis
et al., 1987, Mol. Cell Biol. 7:2435-2443). The DNA probe was made
radioactive with .alpha.-.sup.32 P nucleotide by the random priming
method (Feinberg & Vogelstein, 1983, Anal. Biochem. 132: 6;
Ibid 137:266) to a specific activity of 2.times.10.sup.8 cpm/.mu.g.
Filters were washed in 1.times.SSC, 0.1% SDS for 15 minutes at room
temperature, followed by 15 minutes at 45.degree. C. Filters were
exposed to X-ray film for 24 hours. AS cDNA clones, .lambda.cAS301
and .lambda.cAS305 (FIG. 1A), isolated via heterologous
hybridization to the human AS probe (pH131), were purified via
three rounds of plaque purification and DNA prepared according to
Maniatis et al., 1982, Molecular Cloning A Laboratory Manual, Cold
Spring Harbor Laboratory, pp. 63-67, and 76-85. Restriction
fragments of .lambda.cAS301 (FIG. 1A, 1373 bp SstI/BamHI fragment
"b") and .lambda.cAS305 (FIG. 1A, 423 bp BamHI/EcoRI fragment "c")
were subcloned into pTZ18U or pTZ19U (Genescribe.TM., U.S.
Biochemical Corp., Cleveland, Ohio). For further sequence analysis,
restriction fragments were subcloned into M13mp18 or M13mp19
(Yanisch-Perron, et al., 1985, Gene 33:103-119; Biggin et al.,
1983, Proc. Natl. Acad. Sci. (USA) 80:3963-3965).
6.1.2. SYNTHESIS OF FULL LENGTH AS1 cDNA
cDNA clones corresponding to the 5, end of AS1 mRNA (i.e.
.lambda.cAS907) were synthesized using the following 40 base
oligonucleotide primer complementary to the 5' end of
.lambda.cAS301 (in which "nt" numbers refer to the complementary
nucleotide positions within the full length sequence shown in FIG.
2A): ##STR1##
This 40 base oligonucleotide was annealed with 5 .mu.g pea nodule
polyadenylated RNA. First strand synthesis was performed using
reverse transcriptase according to Verma, 1981, in, The Enzymes:
Nucleic Acids Part A, XIV, Boyer Ed., Acad. Press, N.Y., pp. 87-103
as described (BRL cDNA Synthesis System, Catalog No. 8267SA).
Following second strand synthesis, EcoRI linkers were added
(Maniatis et al., 1982, Molecular Cloning A Laboratory Manual, Cold
Spring Harbor Laboratory, pp. 243-246) and the cDNA fragments were
ligated into Lambda ZAP.RTM.II vector (Stratagene, La Jolla,
Calif.). cDNA inserts were subcloned into M13mp18 and M13mp19 and
analyzed by DNA sequence analysis (Biggin et al., 1983, Proc. Natl.
Acad. Sci. (USA) 80:3963-3965). The full length cDNA clone, pcAS1
(2135bp), encoding AS1 was constructed from the sequential ligation
of the 423 bp BamHI/EcoRI fragment of .lambda.cAS305 (FIG. 1A,
fragment "c"), the 1373 bp SstII/BamHI fragment of .lambda.cAS301
(FIG. 1A, fragment "b"), and the 339 bp EcoRI/SstII fragment of
pcAS907 (FIG. 1A, fragment "a") into the EcoRI site of pTZ18U
(Genescribe.TM., U.S. Biochemical Corp., Cleveland, Ohio) as shown
in FIG. 1A. The resulting clone, designated pTZ18U/cAS1 (or pcAS1)
was deposited with the NRRL as described infra.
6.1.3. ISOLATION OF PLANT AS GENOMIC CLONES
Using the techniques described above, AS1 and AS2 genomic clones
were isolated from Pisum sativum (var. "Feltham First" genomic
library constructed in .lambda. EMBL 3 (Frischauf et al., 1983, J.
Mol. Biol. 170:827-842) using size fractionated partial digests of
Sau3A cleaved leaf DNA amplified in E. coli K803 and screened on
the selective lysogen Q359 (Lycett et al., 1985, Nuc. Acids Res.
13:6733-6743). This genomic library was probed with a portion of
the coding region of the AS1 cDNA (fragment "b" in FIG. 1A). Two
clones were identified and isolated, .mu.gAS1 which hybridized
strongly to the AS1 cDNA probe, and .lambda.gAS2 which hybridized
more weakly to the AS1 cDNA probe (see FIG. 1C). A 3.2 kb
SstI/BamHI fragment of .lambda.gAS1 was subcloned into pTZ18U
resulting in pgAS1.a, and a 5.5 kb BamHI fragment of .lambda.gAS2
was subcloned into pTZ18U resulting in pgAS 2.a as shown in FIG.
1C. Both genomic clones, pgAS1.a and pgAS2.a were deposited with
the NRRL as described infra. Fragments were subcloned into M13mp18
and M13mp19 for sequence analysis shown in FIG. 2C.
6.1.4. ISOLATION OF PLANT AS2 cDNAs
The genomic clone .lambda.gAS2 was used to isolate AS2 cDNA as
follows. A 1.5kb EcoRI/BamHI fragment of AS2 genomic clone,
.lambda.gAS2, containing 3' noncoding region and some of the AS2
coding region (FIG. 1C) was used to screen a pea root cDNA library
(Tingey et al., 1987, EMBO J. 6:1-9) to isolate AS2 cDNA clones.
The pea root cDNA library was used for this purpose because
Northern analyses revealed that in the root, AS2 expression is
higher than that of AS1. The cDNA clone containing the longest
insert, pcAS2-01 (FIG. 1B), was isolated and subcloned into the
EcoRI site of pTZ19U (Genescribe.TM. U.S Biochemical Corp.,
Cleveland, Ohio), resulting in cDNA clone pTZ19U/cAS2-01 (or
pcAS2-01) which was deposited with the NRRL as described infra.
The cDNA clones containing the 5' end of AS2 mRNA were amplified
from pea nodule poly(A).sup.+ RNA by anchored polymerase chain
reaction (A-PCR) technique (Loh et al. 1989, Science 243:217-220).
First strand cDNA was synthesized in a reaction mix containing 50
mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl.sub.2, 50 mM
dithiothreitol, 0.5 mM dNTP, 5 .mu.g nodule poly(A).sup.+ RNA, 200
U M-MLV reverse trancriptase (Bethesda Research Labs, Gaithersburg,
Md.) and 1 .mu.g oligonucleotide FY13
(5'-GGCCGAATTCATACAAATGACCAGGTGGAAAACAC) which includes an EcoRI
site plus sequences complementary to the 5' end of cAS201 (617-641
nt, see FIG. 2B) at 37.degree. C. for 1 hour. The reaction was
stopped by phenol-chloroform extraction and the supernatant was
passed through Linker 6 Quick Spin.TM. Columns (Boehringer Mannheim
Biochemicals, Indianapolis, Ind.) to remove excess linkers. After
ethanol precipitation, the tailing reaction was performed according
to manufacturer's instructions in 50 .mu.l of reaction mix
containing 20 .mu.M of dGTP, 1.times.TdT buffer and 15 U of
terminal deoxynucleotidyl transferase (TdT) (Bethesda Research
Labs) at 37.degree. C. for 30 minutes. After phenol-chloroform
extraction and ethanol precipitation, the tailed cDNAs were
redissolved in 20 .mu.l of water and used as templates in an A-PCR
reaction. The A-PCR reaction was performed with Taq polymerase
(Perkin-Elmer Cetus, Norwalk, Conn.) in 100 .mu.l of a buffer
containing 5 .mu.l of the tailed cDNAs, 0.1 mM of dNTP, 2.5 .mu.g
of AS2 specific primer (FY13) and 2.5 .mu.g of an anchored primer
mix containing a 1:9 ratio of AnC primer
(5'-CAGGTCGACTCTAGAGGATCCCCCCCCCCCCCCC) and An primer
(5'-CAGGTCGACTCTAGAGGATCCC). A program of six cycles of
low-stringency hybridization and amplification (94.degree. C. for
45 seconds followed by annealing at 37.degree. C. for 1 minute and
elongation at 72.degree. C. for 2 minutes) was followed by 24
cycles of high-stringency hybridization and amplification
(94.degree. C. for 45 seconds followed by annealing at 55.degree.
C. for 1 minute and elongation at 72.degree. C. for 2 minutes). The
amplified cDNAs were precipitated by ethanol, digested with EcoRI
and BamHI, then separated on an agarose gel. A predominant DNA
fragment of about 600 bp was recovered from the agarose gel and
ligated into the EcoRI and BamHI sites of pTZ19U (Genescribe). The
ligated DNA was introduced into E. coli XL1 blue. Clones containing
AS2 sequences were isolated and sequenced by the dideoxy
method.
6.1.5. DNA AND RNA ANALYSES
Nuclear DNA from Pisum sativum was analyzed by Southern blot
analysis according to the method described in Tingey et al., 1987,
EMBO J. 6:1-9. Briefly, pea nuclear DNA was digested with SstI,
EcoRI, BamHI or HindIII. The fragments resulting from each digest
were resolved by gel electrophoresis, transferred to nitrocellulose
and probed with a 1373 bp SstI/BamHI .sup.32 P-labeled fragment of
AS1 (FIG. 1A, fragment "b") or an 872 bp BamHI/EcoRI .sup.32
P-labeled fragment of AS2 (FIG. 1B, 3' end of pcAS2-01). The
genomic Southern blot was performed at high stringency (hybridized
at 70.degree. C. and washed at 70.degree. C. in 0.1.times.SSC and
0.1% SDS) such that cAS1 and cAS2 cannot cross-hybridize to each
other.
Northern analyses of mRNA obtained from leaves, roots, nodules or
cotyledons of Pisum sativum were performed according to the method
described by Tingey et al., 1987, EMBO J. 6:1-9. Briefly, the total
RNA or polyadenlyated RNA obtained was denatured, resolved by gel
electrophoresis, transferred to nitrocellulose and probed with a
423 bp BamHI/EcoRI .sup.32 P-labeled fragment of AS1 (FIG. 1A,
fragment "c") or a 224 bp HincII/EcoRI .sup.32 P-labeled fragment
of AS2 (FIG. 1B, 3' end of pcAS2-01), which have specific
activities of about 1-2.times.10.sup.8 c.p.m./.mu.g. The
intensities of gene-specific mRNA were determined by densitometer.
For Northern blot analysis in which poly(A).sup.+ RNA was used, the
quantitations were standardized using .beta.-subunit of
mitochondrial ATPase mRNA as a control. Sizes of mRNAs were
estimated by migration relative to denatured DNA markers.
6.2. RESULTS
6.2.1. ISOLATION OF TWO CLASSES OF PEA AS cDNA CLONES
cDNA clones encoding plant AS were selected from the pea nodule
cDNA library (Tingey et al., 1987, EMBO J. 6:1-9) by hybridization
to a heterologous DNA probe encoding human asparagine synthetase
(Andrulis et al., 1987, Mol. Cell Biol. 7:2435-2443). From 50
positive clones identified out of 2.times.10.sup.5 clones screened,
eight clones (.lambda.cAS301-.lambda.cAS308) were randomly selected
for further analysis. Restriction mapping and nucleotide sequence
analysis of these clones revealed that all eight contained cDNA
inserts which correspond to overlapping portions of a single mRNA
species, AS1 (FIG. 1A). A cDNA clone (.lambda.cAS907) containing
the 5' end of the AS1 mRNA was synthesized using an oligonucleotide
derived from the 5' end of .lambda.cAS301. The restriction maps of
three overlapping cDNA clones which include the entire AS1 coding
region are shown in FIG. 1A. Restriction fragments of the three
overlapping cDNA clones (.lambda.cAS907, .lambda.cAS301, and
.lambda.cAS305) were ligated to form the composite full length 2.2
kb cDNA clone called pcAS1 (FIG. 1A).
A second type of AS coding sequence (AS2) was detected in peas when
a DNA fragment from the coding region of an AS1 cDNA was used to
screen a pea genomic library. cDNA clones encoding the AS2 mRNA
were subsequently isolated from a pea root cDNA library using an
AS2 genomic fragment as a DNA probe. The longest AS2 cDNA clone,
.lambda.cAS201, which contains a 1.5 kb cDNA insert, was selected
for further analysis (FIG. 1B). A cDNA containing the 5' end of the
AS2 mRNA (pcAS801) was synthesized in vitro by anchored polymerase
chain reaction (A-PCR) using an oligonucleotide primer
complementary to the 5' end of .lambda.cAS201 as described above.
The restriction map of the full length cDNA of AS2 (cAS2) was
deduced from the overlapping partial cDNA clones .lambda.cAS201 and
pcAS801.
6.2.2. pcAS1 AND cAS2 REPRESENT HOMOLOGOUS AS mRNAs WHICH ENCODE
DISTINCT AS POLYPEPTIDES
The nucleotide sequences of the full-length AS1 and AS2 cDNAs are
shown in FIG. 2. pcAS1 is 2200 nt long, and starting with the first
in-frame methionine, encodes a protein of 586 amino acids with a
predicted molecular weight of 66.3 kd. The 3' non-coding region of
AS1 cDNA is 333 nt long and contains a poly(A) tail (FIG. 2A). cAS2
is 2002 nt long and encodes a protein of 583 amino acids with a
predicted molecular weight of 65.6 kd. The 3' non-coding region of
cAS2 is 141 nt long and contains a poly(A) tail (FIG. 2B).
As shown in FIG. 3 and Table II, the nucleotide sequence homology
between cDNAs corresponding to AS1 and AS2 mRNA is 81%. This
homology is confined to the protein coding regions of the two
cDNAs, whereas there is no significant homology in the 3'
non-coding regions of these cDNAs. Due to the degeneracy of the
universal code, the cDNAs for AS1 and AS2 are even more homologous
when encoded amino acids are compared 86%). The overall nucleotide
homology between either AS cDNA of pea and the AS cDNA of human is
about 50 to 55% within the coding regions. Neither pea AS cDNA
shares significant homology to the AS gene of E. coli (Nakamura et
al., 1981, Nucleic Acids Res. 9:4669-4676).
TABLE II
__________________________________________________________________________
Percent Homology of Plant AS and Human AS* % Nucleotide Homology %
Amino Acid Homology AS1 AS2 pH131 AS2 AS2 pH131
__________________________________________________________________________
AS1 100% 81% 50% AS1 100% 86% 47% AS2 81% 100% 55% AS2 86% 100% 47%
pH131 -- -- 100% pH131 -- -- 100%
__________________________________________________________________________
*AS1 and AS2 encode plant AS. pH131 is a full length cDNA clone
encoding human AS (Andrulis et al., 1987, supra). Nucleotide and
amino acid homologies were determined using the "fasta" computer
program (Lipman & Pearson, 1985, Science 227:1427; Pearson
& Lipman, 1985, Proc. Natl. Acad Sci. (USA) 85:2444).
That the pea cDNA clones for AS1 and AS2 encode plant AS was
confirmed by a comparison of their encoded amino acids to those
deduced for human AS as shown in FIG. 3. The polypeptides encoded
by the pea AS1 and AS2 cDNAs share an overall homology of 86% at
the amino acid level. A comparison of the pea AS and human AS
polypeptides reveals an overall homology of 47% which extends along
the entire AS polypeptide (Table II). There are several regions of
high local homology (greater than 80%) shared between the pea AS
and human AS polypeptides (amino acid residues 116-128; 218-243;
340-348; 352-360; 392-401; and 486-500 in the pea AS1 protein). In
particular, the first four amino acids of the human AS protein
(Met-Cys-Gly-Ile), which have been shown to be the glutamine
binding site (Heeke and Schuster, 1989, J. Biol. Chem.
264:5503-5509) are perfectly conserved in both the pea AS1 and AS2
proteins. A region of divergence between the pea AS and human AS
proteins occurs at amino acid residues 165-234 of the human AS
protein. This stretch of amino acids is not found in either pea AS1
or AS2 polypeptide and may be the result of gene modification
(deletion or insertion) during evolution of plant versus animal
AS.
6.2.3. AS1 AND AS2 ARE ENCODED BY SINGLE NUCLEAR GENES IN THE PEA
GENOME
Southern blot analysis of nuclear DNA was used to examine the
number of genes encoding AS in Pisum sativum (FIG. 4). Southern
blots probed with a DNA probe from the coding region of pcAS1 as
described, detect a single genomic fragment in each of the four
restriction digests (FIG. 4A, lanes 1-4). Replicate blots probed
with a DNA probe from the coding region of the pcAS201 cDNA reveal
that AS2 is also present as a single gene (FIG. 4B, lanes 5-8). The
results shown in FIG. 4 reveal that in each digestion only a single
genomic DNA restriction fragment hybridizes to each probe. In
addition, the genomic DNA fragments which hybridize to either AS1
or AS2 cDNA probes are distinct. Similar results were obtained with
DNA fragments containing 3' non-coding sequences of pcAS1 or cAS2.
These results indicate that peas contain a single gene for AS1 and
a distinct single gene for AS2.
6.2.4. PHOTOPHOBIC ACCUMULATION OF AS1 mRNA IN LEAVES
Previous biochemical studies have shown that AS enzyme activity
increases when plants are grown in the dark (Joy et al., 1983,
Plant Physiol. 73:165-168). To address whether this increase in AS
enzyme activity reflects an increase in AS gene expression in the
dark, gene-specific probes derived from 3' non-coding regions of
AS1 and AS2 cDNAs were used in Northern blot experiments to detect
AS mRNAs in leaves of plants grown under different light regimes
(FIG. 5). AS1 mRNA (2.2 kb) accumulates to high levels in leaves of
mature dark-adapted green plants (FIG. 5A, lanes 2 and 3). However,
when these plants are transferred to continuous white light, the
steady-state levels of AS1 mRNA decrease dramatically to almost
undetectable levels (FIG. 5A, lanes 4 and 5). In mature plants,
both the dark-induced and light-repressed accumulation of AS1 mRNA
can be detected 6 hours after changing the light conditions (FIG.
5A, lanes 2 and 4 respectively). The steady-state levels of AS1
mRNA in leaves of dark-adapted plants (FIG. 5A, lane 3) are 30-fold
higher than the AS1 mRNA levels present in leaves of light-grown
plants (FIG. 5A, lane 5). As a control, mRNA for cytosolic GS (1.4
kb) monitored on the same blot revealed no dramatic changes in mRNA
levels in response to the light treatments. AS2 mRNA levels were
also detected on replicate blots with a DNA probe from the 3'
non-coding region of cAS2. These experiments revealed that AS2 mRNA
(2.2 kb) is present at much lower levels than AS1 mRNA in leaves of
dark-grown plants and is undetectable in leaves of grown
plants.
Northern blots were also performed on RNA isolated from plants at
various developmental stages which were grown in continuous white
light (FIG. 5B, lanes 1, 3, 5 and 7) and then transferred to the
dark (FIG. 5B, lanes 2, 4, 6 and 8). The results of these
experiments reveal that the dark-induced accumulation of AS1 occurs
in plants of all developmental stages but is most dramatic in
mature plants. The dark-induced increase of AS1 mRNA varies from
5-fold in 10 day old plants (FIG. 5B, compare lanes 1 and 2) to
greater than 20-fold in 31 day old plants (FIG. 5B, compare lanes 7
and 8). As a control, Northern blots reprobed with a DNA probe
encoding a cytosolic form of glutamine synthetase, GS (Tingey et
al., 1988, J. Biol. Chem. 263:9651-9657, which is incorporated by
reference herein in its entirety) reveal that the mRNA for
cytosolic GS (1.4 kb) is relatively unaffected by the different
light treatments (FIG. 5B, lower band).
AS1 mRNA (2.2 kb) is expressed at high levels in leaves of
dark-grown or dark-adapted plants (FIG. 5C, lane 1, and FIG. 5D
lane 2, respectively). AS1 mRNA levels decrease dramatically when
these plants are transferred to continuous white light (FIG. 5C,
lane 2, and FIG. 5D, lane 3). This "photophobic" nature of AS1 mRNA
accumulation is also evident in mature light-grown pea plants (FIG.
5D lane 1). The photophobic accumulation of AS1 mRNA in the dark is
in direct contrast to the light-induced accumulation of the 1.5 kb
mRNA for chloroplast GS2 (Tingey et al., 1988, J. Biol. Chem.
263:9651-9657); see FIGS. 5C and 5D, lower molecular weight signal.
AS2 mRNA is present at relatively low levels in leaves of dark- or
light-grown plants. It is noteworthy that the photophobic
accumulation of AS1 mRNA in leaves of plants transferred to
darkness occurs in plants at various stages of development (FIG.
5B). The mRNA for cytosolic GS (1.4 kb) is unaffected by the light
treatments (FIG. 5B, lower molecular weight signal).
In order to determine whether the plant photoreceptor phytochrome
is involved in mediating the dark-induced expression of AS1 gene in
peas, AS1 mRNA was examined in etiolated plants treated with light
regimes known to activate or inactivate phytochrome (FIG. 5E).
While AS1 mRNA accumulates to high levels in etiolated plants (FIG.
5E, lane 1) the levels of AS1 mRNA decrease dramatically in plants
treated with a red-light pulse (FIG. 5E, lane 2). The repression of
AS1 expression by red light is partially reversed by a subsequent
pulse of far-red light (FIG. 5E, lane 3). The effects of red and
far-red light pulses on accumulation of AS1 mRNA were detected
within 3 hours after light treatment. These results show that the
dark-induced accumulation of AS1 mRNA is mediated, at least in
part, through the chromophore phytochrome.
6.2.5. BOTH AS1 AND AS2 mRNAs ARE EXPRESSED AT HIGH LEVELS DURING
DEVELOPMENTAL CONTEXTS INVOLVING INCREASED NITROGEN TRANSPORT
To determine whether the level of AS mRNA increases in contexts
where large amounts of asparagine are synthesized for nitrogen
transport, the steady-state levels of AS1 and AS2 mRNAs were
monitored in nitrogen-fixing root nodules of peas and in cotyledons
of germinating pea seedlings (FIG. 6). Asparagine serves as a major
nitrogen transport amino acid during germination. The results of
the gene-specific Northern blots for AS reveals that both AS1 and
AS2 mRNAs accumulate to high levels in cotyledons of germinating
pea seedlings (FIG. 6A). While AS1 mRNA can be detected after 10
days of germination (FIG. 6A, lane 5' panel), AS2 mRNA is detected
earlier (4-6 days of germination) (FIG. 6A, lanes 2 and 3' lower
panel). There is a greater than 20-fold increase of both AS1 and
AS2 mRNAs in cotyledons during germination time course (FIG. 6A,
compare lanes 2 and 7). The same Northern blot reprobed with DNA
probe for a cytosolic form of glutamine synthetase, GS (Tingey et
al., 1988, J. Biol. Chem. 263:9651-9657), reveals that mRNA for
cytosolic GS accumulates earlier (2-4 days) than either AS
mRNA.
AS mRNA levels were also examined in nitrogen-fixing root nodules
of pea where asparagine serves as a major compound for nitrogen
transport from nodules to the rest of the plant. RNA from
nitrogen-fixing root nodules and roots of uninfected plants were
probed in Northern blot experiments with gene-specific AS probes
(FIG. 6B). These experiments reveal that both AS1 and AS2 mRNAs
accumulate to very high levels in nitrogen-fixing nodules (FIG. 6B,
lane 2) compared to uninfected roots (FIG. 6B, lane 1). The
induction of AS1 mRNA in nodules compared to roots is 20-fold while
that of AS2 mRNAs is only 5-fold. The lower fold induction of AS2
mRNA may reflect the higher basal levels present in uninfected
roots (FIG. 6B, lane 1). As a control, the Northern blot was
reprobed with a DNA probe for the .beta.-subunit of the
mitochondrial ATPase (Boutry and Chua, 1985, EMBO J. 4:2159-2165)
which is expressed at equal levels in roots of uninfected plants
and nitrogen-fixing nodules (FIG. 6B, lower panel).
6.3. DISCUSSION
While asparagine is an important nitrogen transport amino acid in
higher plants, the enyzme involved in its synthesis is poorly
characterized to date due to enzyme instability in vitro. In the
examples described, supra, plant AS cDNAs were cloned using a
heterologous DNA probe encoding human AS. Two classes of AS cDNAs
(AS1 and AS2) that encode homologous but distinct AS proteins were
obtained from pea cDNA libraries. The homologies between the pea
AS1 and AS2 cDNAs are 81 and 86% at nucleotide and amino acid
levels respectively. Full-length cDNAs for AS1 and AS2 of pea were
shown to encode proteins whose sizes and amino acid sequences are
in excellent agreement with that deduced for the human AS protein.
The pea AS1 and AS2 cDNAs and human AS cDNA share an overall
nucleotide homology of 50-55% along their entire coding sequence.
Regions that are highly conserved between the pea AS and human AS
polypeptides (greater than 80% at amino acid level) may likely
include important sites for enzyme activity. For example, the first
four amino acids in the human AS protein (Met-Cys-Gly-Ile), which
have been shown to be the glutamine-binding site and important for
enzyme activity (Andrulis et al 1987, Mol. Cell. Biol. 7:2435-2443;
Heeke and Schuster, 1989, J. Biol. Chem. 264:5503-5509) are
perfectly conserved in both the pea AS1 and AS2 proteins. The
degree of sequence homology between the pea AS and human AS
proteins supports the conclusion that the full length AS1 and AS2
cDNAs encode glutamine-dependent AS of peas.
The significance of two homologous but distinct AS polypeptides in
plants is intriguing. The AS1 and AS2 cDNAs of pea may encode two
distinct subunits of a single AS holoenzyme (heterologous
holoenzyme); or each subunit may assemble into a separate AS
holoenzyme (homologous holoenzyme of either AS1 or AS2 subunits).
These two possibilities are not mutually exclusive. Partially
purified plant AS enzyme preparations have been shown to utilize
glutamine as a preferred substrate; however, ammonia can also be
used as a substrate in the same preparations albeit with higher
K.sub.m values (Scott et al., 1976, Nature 263:703-705; Huber et
al., 1984, Plant Physiol. 74:605-610; 1985, Plant Sci. 42:9-17 .
The existence of glutamine binding sites at the amino terminus of
the pea AS1 and AS2 proteins implies that both AS1 and AS2 genes
encode glutamine-dependent forms of AS. It is possible, however,
that these AS enzymes are able to utilize ammonia as a substrate in
vivo under conditions of ammonia excess. It is interesting to note
that glutamine versus ammonia-dependent forms of AS are encoded by
separated genes in E. coli (Felton et al., 1980, J. Bacteriol. 142
221-228; Humbert et al., 1980, J. Bacteriol. 142:212-220) and yeast
(Jones, 1978, J. Bacteriol. 134:200-207; Ramos et al., 1980, Euro.
J. Biochem. 108:373-377). Therefore, we cannot exclude the
possibility that plants might contain another distinct AS gene for
ammonia-dependent form of AS. Previous biochemical studies have
also shown that AS activity can be detected in both soluble and
proplastid fractions of nitrogen-fixing nodules of soybean (Boland
et al., 1982, Planta 155:45-57). The proteins encoded by AS1 and
AS2 cDNAs of pea are most likely cytosolic AS since neither of them
contains a transit peptide. It is possible that peas contain
another distinct gene for plastid AS.
Northern blot analysis has revealed that the steady-state levels of
AS1 and AS2 mRNAs parallel asparagine synthesis in various
developmental contexts. For example, previous physiological studies
have shown that asparagine is the major nitrogen transport amino
acid in plants grown in the dark (Urquhart et al., 1981, Plant
Physiol. 68:750-754) and that AS activity can be enhanced by dark
treatment (Joy et al. 1983, Plant Physiol. 73:165-168). The
experiments described herein demonstrate that in peas the increase
of AS activity in the dark is due, at least in part, to an increase
in the steady-state levels of AS1 mRNA. This dark-induced
accumulation of AS1 mRNA occurs in leaves of both etiolated
seedlings and in mature dark-adapted green plants. Moreover, the
magnitude of dark-induced AS1 mRNA accumulation increases
significantly during plant development. Kinetic experiments reveal
that both dark-induced and light-repressed changes in AS1 mRNA
levels can be detected within 3 hours in etiolated seedlings (FIG.
5E) and mature plants after changing the light/dark conditions.
Thus, the dark-induced accumulation of AS1 mRNA is physiologically
significant for plants grown in a short dark period (e.g. at
night).
The dark-induced accumulation of the AS1 mRNA classifies the AS1
gene with other genes that are negatively regulated by light such
as phytochrome (Otto et al., 1984, Plant Cell Physiol.
25:1579-1584; Lissemore et al., 1988, Mol. Cell. Biol. 8:4840-4850;
Kay et al. 1989, Plant Cell 1:357-360), protochlorophyllide
reductase (Mosinger et al., 1985, Eur. J. Biochem. 147:137-142) and
an unidentified mRNA found in Lemna (Okubara et al., 1988, Plant
Mol. Biol. 11:673-681). As shown for phytochrome (Lissemore et al.,
1988; Kay et al., 1989) and protochlorophyllide reductase genes
(Mosinger et al., 1985), the repression of AS1 mRNA accumulation in
the light is a phytochrome-mediated response. We have determined
that the dark-induced (or light-repressed) expression of AS1
reflects a transcriptional response as has also been shown for
phytochrome (Lissemore and Quail, 1988; Kay et al., 1989) and
protochlorophyllide reductase (Mosinger et al., 1985). The rapid
changes in levels of AS1 mRNA suggest that a post-transcriptional
response (e.g., mRNA stability) may also be involved as has been
shown for another dark-induced gene of unknown function (Okubara et
al., 1988). In direct contrast to the dark-induced accumulation of
AS1 mRNA in leaves, the mRNA for the chloroplast form of glutamine
synthetase (GS2) accumulates in the light in a phytochrome-mediated
response (Tingey et al., 1988, J. Biol. Chem. 263:9651-9657).
Parallel molecular studies on the mechanisms for dark-induced
accumulation of AS1 mRNA and light-induced accumulation of GS2 mRNA
will uncover how two genes encoding nitrogen metabolic enzymes
along a common pathway are regulated by light via phytochrome in
opposite fashions.
Previous biochemical studies have revealed high levels of AS
activity in two developmental contexts where large amounts of
asparagine are synthesized for nitrogen transport: in cotyledons of
germinating seedlings (Capdevila et al., 1977, Plant Physiol.
59:268-273; Dilworth et al., 1978, Plant Physiol. 61:698-702; Kern
et al., 1978, plant Physiol. 62:815-891) and in nitrogen-fixing
root nodules (Scott et al., 1976, Nature 263:703-705; Reynolds et
al., 1982, Physiol. Plant 55:255-260). Previous studies also showed
that actinomycin D treatment abolished the induction of AS activity
in cotyledons of germinating cotton seedlings, indicating that AS
expression in cotyledons is regulated at the transcriptional level
(Capdevila et al., 1977; Dilworth et al., 1978 . Consistent with
those findings, we have shown that the accumulation of both AS1 and
AS2 mRNAs are induced to high levels in cotyledons of germinating
seedlings. Comparative studies of AS mRNAs and GS mRNAs in this
context show that the steady-state levels of mRNA for cytosolic GS
accumulate earlier than those of both AS mRNAs in cotyledons of
germinating seedlings. These results suggest that glutamine
synthesized by GS may act as a metabolic signal to induce AS gene
expression in this developmental context. The mRNAs of AS1 and AS2
also accumulate to very high levels in root nodules of peas in a
parallel fashion with cytosolic GS mRNA. The accumulation of AS1
and AS2 mRNA in nitrogen-fixing nodules may be the result of
factors produced by the process of nodulation or/and by metabolic
factor(s) such as ammonia or glutamine production in nodules.
Southern blot analysis of genomic DNA reveals that the gene family
for AS in peas is composed of at least two genes, AS1 and AS2,
which encode homologous but distinct gene products. Expression
studies show that AS1 and AS2 genes share some similarities in
expression patterns (e.g. induced accumulation of mRNA in
cotyledons and nodules); however, they have distinct organ-specific
patterns of expression. AS1 mRNA accumulates to higher levels in
leaves compared to AS2, while AS2 mRNA accumulates to higher levels
in roots than AS1. In this respect, the AS gene family resembles
the GS gene family where members of a gene family may be
differentially regulated by distinct factors which modulate
expression of individual genes in specific contexts during
development.
7. EXAMPLE: THE AS PROMOTER
The subsections below describe the AS promoter sequence and its
induction by dark.
7.1. DARK-INDUCED ACCUMULATION OF AS mRNA OCCURS IN ALL ORGANS
TESTED
The results described in Section 6, supra, demonstrate that the AS1
mRNA accumulates within 6 hours of dark-treatment and that the
photoreceptor phytochrome mediates the dark-induced accumulation of
AS1 mRNA. In the experiments described herein, we have examined the
effect of dark-treatment on AS gene expression in other plant
organs. The steady-state levels of AS1 and AS2 mRNA were detected
by gene specific Northern blot analysis in leaves, stems, and roots
of light-grown (FIG. 7, lanes 1, 3, 5) or dark-adapted (FIG. 7,
lanes 2, 4, 6) pea plants. These results show that AS1 mRNA
accumulates to very high levels in all organs of dark-treated
plants. The accumulation of AS2 mRNA is also induced by
dark-treatment in leaves and stems. However, the steady-state
levels of AS2 mRNA in dark-adapted plants was lower than that of
AS1 mRNA. Interestingly, the steady-state levels of AS2 mRNA do not
increase in roots after dark-treatment. This may be due to higher
steady-state levels of AS2 mRNA in light-grown roots compared to
AS1.
7.2. THE DARK-INDUCED EXPRESSION OF AS1 GENE IS NOT REGULATED BY
CIRCADIAN RHYTHM
Gene-specific Northern blot analysis on the total RNA isolated from
leaves collected at different time points of a day with variant
light/dark conditions were performed to examine whether circadian
rhythm is involved in the regulation of AS1 gene expression (FIG.
8). FIG. 8A shows that the accumulation of AS1 mRNA can be detected
within 5 hours in the darkness and reach peak levels after 8 hours
in the darkness. The decrease of AS1 mRNA level can be detected
within 3 hours in the light; after 6 hours in the light the AS1
mRNA almost reach the minimum level. In the condition of extended
darkness, the AS1 mRNA levels were kept at a very high level (FIG.
8B). On the contrary, the extended light condition maintained the
AS1 mRNA at basal low levels (FIG. 8C). These results reveal that
circadian rhythm is not involved in the dark-induced expression of
AS1 gene. As a control, the mRNA levels of cytosolic GS were
detected.
7.3. THE DARK-INDUCED EXPRESSION OF AS GENES IN PEA LEAVES IS
REGULATED AT THE LEVEL OF TRANSCRIPTION
Nuclear run-on assays were performed to determine whether
dark-treatment increases the transcription rate of AS gene (FIG.
9). These run-on experiments demonstrate that the AS1 gene is
transcribed at a high level in leaves of dark-grown peas (FIG. 9,
panel 2D). For AS2, the transcription rate is significantly lower
than that of AS1 in leaves of dark-grown peas (FIG. 9, panel 3D).
As a control, the transcription of genes for chloroplast GS and
cytosolic GS were also monitored (FIG. 9, panels 4, 5). After the
pea plants were transferred to continuous light for 6 hours, the
transcription rate of AS1 decreases dramatically and the
transcription rate of AS2 is also lower in light grown leaves (FIG.
9, panels 2L, 3L). These results show that while both AS1 and AS2
genes are preferentially transcribed in the dark, the AS1 gene is
transcribed at a higher level in dark-grown leaves. As a control,
we demonstrated that the transcription rate of the nuclear gene for
chloroplast GS increases after light-treatment, and the
transcription rate of cytosolic GS is relatively unchanged in
different dark/light conditions (FIG. 9, panels 4, 5). These
results demonstrate that the dark-induced expression of both AS1
and AS2 genes is regulated at the transcriptional level.
7.4. THE 569 BASE PAIR FRAGMENT OF THE AS1 PROMOTER IS SUFFICIENT
TO DRIVE GUS EXPRESSION IN TRANSGENIC PLANTS
FIG. 10 shows the AS1 promoter sequence with nucleotides numbered
relative to the transcription start site. Four AS1 promoter-GUS
transcriptional fusions containing different lengths of the AS1
promoter fused to GUS were made and introduced into tobacco plants
(FIG. 11). GUS activity was analyzed in leaves of dark-adapted
transgenic plants to determine the expression of the GUS gene
driven by AS1 promoter. GUS activity was detected in leaves of five
dark-grown transgenic tobacco plants. Among them, two contain the
pBI-AS1001 construct, and three contain the pBI-AS1003 construct
(FIG. 11). Four out of these five transgenic tobacco express GUS
specifically in phloem cells. We have also found a phloem-specific
expression pattern of cytosolic GS (Jefferson, 1987, Plant Mol.
Bio. Reporter 5:387-405). These results strongly suggest that the
AS1 gene is specifically expressed in phloem cells, and that the
569 base-pair fragment containing nucleotides -558 to +11 of the
AS1 promoter is sufficient to drive the phloem-specific expression
of GUS in leaves of dark-grown plants.
7.5. AS1 AND AS2 GENES CONTAIN CONSERVED SEQUENCE IN THEIR
PROMOTERS
The promoter sequence of the AS2 gene was determined by sequencing
serial deletion clones of gAS2a fragment (FIG. 12). Since the
sequence from nucleotides -558 to +11 of AS1 gene is sufficient to
drive the phloem-specific expression in the dark (FIG. 11), this
portion of the AS1 promoter was compared with the entire AS2
promoter sequence shown in FIG. 12. FIG. 13 shows sequences
conserved between AS1 and AS2 promoters as determined by the
computer program DNASIS.
8. DEPOSIT OF MICROORGANISMS
The following microorganisms have been deposited with the
Agricultural Research Culture Collection, Northern Regional
Research Center (NRRL) and have been assigned the following
accession numbers:
______________________________________ Microorganism Plasmid
Accession No. ______________________________________ Escherichia
coli XL1 pTZ18U/cAS1 B-18487 Escherichia coli XL1 pTZ19U/cAS201
B-18486 Escherichia coli XL1 pTZ18U/gAS1.a B-18492 Escherichia coli
XL1 pTZ18U/gAS2.a B-18493 Escherichia coli XL1 pTZ19U/cAS801
B-18649 ______________________________________
The present invention is not to be limited in scope by the
microorganisms deposited since the deposited embodiments are
intended as illustrations of single aspects of the invention and
any microorganisms which are functionally equivalent are within the
scope of the invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
It is also to be understood that all base pair sizes given for
nucleotides are approximate and are used for purposes of
description.
* * * * *